Androgenetic haploid embryonic stem cell, and preparation method and use thereof

ABSTRACT

The present invention relates to an AG-haESCs in which H19 DMR and IG-DMR are knocked out, a method for preparing the AG-haESCs, and use of the AG-haESCs in constructing a genetically modified semi-cloned animal and a library of a genetically modified semi-cloned animal. The AG-haESCs is capable of obtaining characteristics resembling a round spermatid, and upon injection into an oocyte, a viable SC mouse is stably obtained. The present invention is capable of being effectively used in multi-gene genetic manipulation, advancing the acquisition of animals with multiple genetic modifications.

BACKGROUND OF THE PRESENT INVENTION Field of Invention

The present invention relates to biotechnologies, and specifically to anandrogenetic haploid embryonic stem cell (AG-haESC), and a preparationmethod and use thereof.

Description of Related Arts

Genome-wide recessive genetic screening is an extremely effective andpowerful method of identifying the functions of a gene involved in agiven biological process. This strategy has been successfully used inlower organisms such as Saccharomyces cerevisiae and nematodes. Inmammals, this functional screening is exceptionally difficult due to thediploidy of the genome (Shi, L., Yang, H., and Li, J. (2012). Haploidembryonic stem cells: an ideal tool for mammalian genetic analyses.Protein & cell 3, 806-810). The means of targeting RNA interference(RNAi) at mRNA level has now become an optimal solution for genome-wideloss-of-function genetic screening in mammalian cells. However, thismethod often cannot effectively inhibit the gene expression, while thereis an off-target effect (Kaelin, W. G., Jr. (2012). Molecular biology.Use and abuse of RNAi to study mammalian gene function. Science 337,421-422). Recently, the bacterial-derived CRISPR-Cas9 system has beensuccessfully used in the screening of genetic defects of the mouse andhuman at cellular levels. However, CRISPR-Cas9-mediated genome-widescreening is only used at cellular level, which limits the studyexclusively to phenotyping at cellular level. In this regard, ascurrently done with lower organisms such as yeast and nematodes, it isimportant to achieve efficient, large-scale loss-of-function screeningof a broader range of biological processes in mammalian systems.

The obtaining of mammalian haploid embryonic stem cells (haESCs)(Elling, U., Taubenschmid, J., Wirnsberger, G., O'Malley, R., Demers, S.P., Vanhaelen, Q., Shukalyuk, A. I., Schmauss, G., Schramek, D.,Schnuetgen, F., et al. (2011). Forward and reverse genetics throughderivation of haploid mouse embryonic stem cells. Cell Stem Cell 9,563-574; and Leeb, M., and Wutz, A. (2011). Derivation of haploidembryonic stem cells from mouse embryos. Nature 479, 131-134) provides adesirable tool for genetic analysis. The androgenetic haploid embryonicstem cells (AG-haESCs) have a whole genome derived from spermatid, withwhich the full development of a reconstructed embryo can be achieved byintracytoplasmic AG-haESCs injection (ICAHCI) into mature MII oocytes,thereby obtaining a viable animal subject that is referred to assemi-cloned animal. It is inferred that if semi-cloned mice can beproduced with AG-haESCs efficiently and stably by ICAHCI, importantgenes involved in a particular developmental process can be screened outby using AG-haESCs as fertilizing vectors to carry genome-wideCRISPR-Cas9 knockout library.

However, previous studies have shown that the birth rate of viable,semi-cloned mice is very low (where the birth rate is 4.5% forhalf-cloned mice and 1% for semi-cloned rats), while approximately 50%of the semi-cloned mice exhibit a phenotype of retarded developmental,and are died shortly after birth. Furthermore, with the long-termculture of AG-haESCs, the overall birth rate of semi-cloned micedeclines rapidly, especially for the additional culture resulting fromgenetic manipulation.

One of the possible reasons is the abnormal expression of imprintedgenes. These imprinted genes that are expressed in aparent-of-origin-specific manner are considered as an important barrierto the development of uniparental embryos, so that the normal growth anddevelopment of embryos require both the maternal and paternal genomes.At present, about 150 imprinted genes have been identified in mice, mostof which are located in a large cluster of genes and are regulated bydifferentially methylated regions (DMRs) (Bartolomei, M. S. (2009).Genomic imprinting: employing and avoiding epigenetic processes. Genes &development 23, 2124-2133). Consistent with this hypothesis, studieshave shown that the imprinting of differentially methylated region (DMR)in which the imprinted gene H19 with inhibited paternal expressionlocated is consistently abnormally erased in both AG-haESCs andgrowth-arrested, semi-cloned mice (Yang, H., Shi, L., Wang, B. A.,Liang, D., Zhong, C., Liu, W., Nie, Y., Liu, J., Zhao, J., Gao, X., etal. (2012). Generation of genetically modified mice by oocyte injectionof androgenetic haploid embryonic stem cells. Cell 149, 605-617). Thematernally expressed H19 gene is adjacent to the paternally expressedIgf2, and both of them are regulated by the same DMR. This DMR isamenable to DNA methylation in the paternal allele, and as aCTCF-dependent insulator, permits the expression of the maternal alleleonly. The methylation state of this DMR determines whether H19 (whereH19 is expressed, DMR is demethylated, and the insulator is activated)or Igf2 (where Igf2 is expressed, DMR is methylated, and the insulatoris deactivated) is expressed. Interestingly, knockout of the H19 gene orits DMR does not lead to any severe phenotype in mice (Leighton, P. A.,Ingram, R. S., Eggenschwiler, J., Efstratiadis, A., and Tilghman, S. M.(1995). Disruption of imprinting caused by deletion of the H19 generegion in mice. Nature 375, 34-39; and Thorvaldsen, J. L., Mann, M. R.,Nwoko, O., Duran, K. L., and Bartolomei, M. S. (2002). Analysis ofsequence upstream of the endogenous HI9 gene reveals elements bothessential and dispensable for imprinting. Molecular and cellular biology22, 2450-2462). At present, there are related researches onreconstructed embryos obtained by using fully mature oocytes andgenetically modified immature oocytes (Kono, T., Obata, Y., Wu, Q.,Niwa, K., Ono, Y., Yamamoto, Y., Park, E. S., Seo, J. S., and Ogawa, H.(2004). Birth of parthenogenetic mice that can develop to adulthood.Nature 428, 860-864; and Kawahara, M., Wu, Q., Takahashi, N., Morita,S., Yamada, K., Ito, M., Ferguson-Smith, A C., and Kono, T. (2007).High-frequency generation of viable mice from engineered bi-matemalembryos. Nature biotechnology 25, 1045-1050). However, the immatureoocytes cannot be cultured and expanded in vitro, so the in-vitrogenetic modification cannot be realized; and the technique of obtainingreconstructed embryos from fully-matured oocytes and geneticallymodified immature oocytes is difficult in operation, so the applicationvalue is not high.

SUMMARY OF THE PRESENT INVENTION

In view of the disadvantage of low birth rate of semi-cloned animals inthe prior art, an object of the present invention is to provide anandrogenetic haploid embryonic stem cell (AG-haESC) that can enhance thebirth rate of semi-cloned animals and use thereof.

The most possible cause of low birth rate is the occurrence of abnormalimprinting status of the androgenetic haploid. It is surprisingly foundthrough experiments that characteristics of the AG-haESCs resemblingthose of a round spermatid can be established by knocking out thedifferentially methylated regions (DMRs) of two imprinted clustersH19-Igf2 and Dlk1-Dio3. Such AG-haESCs with two DMRs knocked out isdesignated as DKO-AG-haESCs. With the DKO-AG-haESCs, semi-cloned micecan be obtained effectively (with a birth rate of SC mice of about 20%),and semi-cloned animals with multiple genetic modifications can still beproduced stably after in-vitro genetic manipulation of the AG-haESCs.Unexpectedly, a large number of mutant animals can be effectivelyobtained in one step simply by transfecting the DKO-AG-haESCs withstable expression of sgRNA library and Cas9 in vitro at the cellularlevel. These experimental results show that DKO-AG-haESCs can be used asan intermediate to achieve gene mutation at the individual level bycarrying the sgRNA library and therefore can be further used in genemutation-based large-scale screening at individual animal level.

A first aspect of the present invention provides an AG-haESC, in whichH19 DMR and IG-DMR genes are knocked out.

A second aspect of the present invention provides a method for preparingthe AG-haESC, which comprises knocking out H19 DMR and IG-DMR from anAG-haESC, to obtain the above mentioned AG-haESC.

A third aspect of the present invention provides use of the AG-haESC inconstructing a genetically modified semi-cloned animal.

A fourth aspect of the present invention provides a method forconstructing a genetically modified semi-cloned animal, which comprisescombining an AG-haESC in which H19 DMR and IG-DMR genes are both knockedout with an oocyte to obtain a semi-cloned embryo, and incubating thesemi-cloned embryo, to obtain a semi-cloned animal.

A fifth aspect of the present invention provides a genetically modifiedanimal, which is constructed according to the above method, or is asexually reproduced offspring of a semi-cloned animal constructedaccording to the above method.

A sixth aspect of the present invention provides a method forconstructing a genetically modified semi-cloned animal library with theAG-haESCs according to the present invention and a lentiviral sgRNAlibrary.

A seventh aspect of the present invention provides a geneticallymodified semi-cloned animal library constructed by using the abovemethod.

The present invention has the following beneficial effects.

(1) Existing studies show that semi-cloned animals can be obtained fromAG-haESCs established with haploid blastocyst by ICAHCI. However,AG-haESCs cannot produce viable SC animals after long-term subculture,especially after in vitro genetic manipulation, probably due to the lossof male imprinted genes. It is found in the present invention that theAG-haESCs is capable of obtaining characteristics resembling a roundspermatid by knocking out H19 and IG-DMR. Upon injection into an oocyte,a viable SC mouse is stably obtained with AG-haESCs at a rate of about20%, which is about 10 times of the rate obtained with earliergeneration of WT AG-haESCs (Yang et al., 2012). Importantly, it is alsofound in the present invention that DKO-AG-haESCs can be effectivelyused for multi-gene genetic manipulation, and SC mice with multiplegenetic modifications can be further obtained by ICAHCI. Moreimportantly, by incorporating with a sgRNA library, DKO-AG-haESCs can beeffectively used in one-step generation of heterozygous and homozygousmutant mice, providing further evidence for gene knockout-basedscreening at animal individual level.

(2) DKO-AG-haESCs are advantageous over the existing methods forobtaining genetically modified animals in the following aspects.Firstly, existing studies report the one-step production of transgenicmice or mice with endogenous genetic mutations by direct injection ofCas9 mRNA and sgRNA into fertilized eggs. This method demonstrates thestrong potential of CRISPR-Cas9 technique in the production ofgenetically modified animals. However, there is an extremely high rateof chimerism observed in the genetically manipulated animals so far,which greatly increases the difficulty of phenotyping in F1 generationof animals. In contrast, DKO-AG-haESCs-mediated gene editing technologyprovides a unique set of system for well phenotyping and screeninggenetically modified DKO-AG-haESCs, to ensure that the resulting SCanimals exhibit the expected genetic traits, so the occurrence ofchimerism can be largely avoided. Secondly, genetically modified miceobtained by conventionally injecting diploid embryonic stem cells intothe blastocysts need to experience the germline transmission of chimericmice. However, the step of germline transmission is often verytime-consuming. More seriously, the diploid embryonic stem cells withmultiple genetic modifications may exhibit trait segregation in theiroffspring during germline transmission. Only a small percentage ofoffspring may contain all the desired genetic modifications. Incontrast, F0 mice with multiple genetic modifications can be obtainedwith DKO-AG-haESCs in one step. Thirdly, fully ESC-derived mice can beobtained in one step by injecting diploid embryonic stem cells into thetetraploid blastocysts (also known as tetraploid complementationtechnique). This method is also widely used and is the most stringentstandard for testing the pluripotency of reprogrammed cells such asinduced pluripotent stem cells (iPSCs) and nuclear transfer embryonicstem cells (ntESCs). However, the birth rate of the embryonic stem cell(ESC)- or iPSC-derived animals is very low, probably due to theepigenetic instability of diploid embryonic stem cells. In contrast,animals can still be stably and efficiently obtained with DKO-AG-haESCsafter long-term in vitro culture, especially genetic manipulation.Fourthly, the ICAHCI technology is similar to ROSI that is a sophiscatedtechnology for various mammals, including non-human primates and human.Cynomolgus monkey-derived haploid embryonic stem cell line has now beensuccessfully established and the ICAHCI technology may be used in thenear future to efficiently and rapidly obtain genetically modifiednon-human primates. Lastly, a large number of genetically mutatedanimals can be obtained with DKO-AG-haESCs by incorporating with a sgRNAlibrary. Obviously, this method is simpler and less labor-intensive thandirect injection of plasmids or mRNAs into embryos for the large-scaleproduction of mutant animals, since the DKO-AG-haESC line, which carriesconstantly expressed Cas9 and sgRNA library, can repeatedly serve as adonor for injection, to produce various genetically mutated animalscontinuously and effectively. These various mutant animals can be easilyand quickly identified for the sgRNA insertion (which is exactly abarcode for a mutant animal) by PCR using a universal pair of primers.In contrast, the method of injecting plasmids or mRNAs directly intoembryos requires separate preparation of sgRNAs for each individualinjection. The mutant animals obtained need to be housed separately, andthe subsequent identification requires the use of corresponding primersfor a particular gene. It is conceivable that some of the differentiallyexpressed genes obtained from an appropriate sgRNA library, such asthose obtained by high-throughput analysis at the cellular level, can beclassified into a sub-library of sgRNAs that may be involved in aparticular stage of development. By using the DKO-AG-haESC solution,important genes related to this development process can be rapidly andeffectively screened out at the individual level.

(3) Production of DKO-AG-haESC-mediated SC animals is an effective andsimple way to generate animal models that carry genetic modificationssuch as multiple genetic mutations or multiple gene knockins in a genefamily. Moreover, by incorporating with a sgRNA library, a large numberof genetically mutated animals can be obtained in one step withDKO-AG-haESCs. Although the underlying mechanism that DKO-AG-haESCs isso effective in the production of SC animals is still unknown, thisapproach may be able to facilitate the deeper exploration for thedevelopmental process and complex diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a view schematically showing the production of SC mice byintracytoplasmic AG-haESCs injection (ICAHCI) of AG-haESCs bearing 2 DMRknockouts.

FIG. 1B shows androgenetic haploid embryos produced by injection ofH19^(Δ) ^(DMR) spermatids into enucleated MII oocytes.

FIG. 1C shows one of the AG-haESC lines established with an androgenetichaploid blastocyst.

FIG. 1D shows H19^(Δ) ^(DMR) AG-haESCs (H19^(Δ) ^(DMR) -AGH-1)established by multiple FACS enrichments of haploid cells.

FIG. 1E shows SC mice produced with H19^(Δ) ^(DMR) -AGH-1 (p8) byICAHCI.

FIG. 1F is a view schematically showing an IG-DMR knockout sgRNAsequence.

FIG. 1G shows production of H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGH-1 cellline. Left panel: collected mCherry positive cells plated in a Petridish. Right panel: established H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGH cellline (H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGH-4).

FIG. 1H shows SC mice produced with H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGU-4(p29) by ICAHCI.

Fig. S1A shows genotyping of H19^(Δ) ^(DMR) -AGH cell line.

Fig. S1B shows that 80% of the embryos reconstructed with H19^(Δ) ^(DMR)-AGH cells reach 2-cell embryos, which is similar to the developmentalrate obtained upon round spermatid injection (ROSI).

Fig. S1C shows SC mice produced with H19^(Δ) ^(DMR) -AGH-1, 2, 3 celllines by ICAHCI, in which asterisk represents growth-arrested SC mice.

Fig. S1D shows genotyping of H19^(Δ) ^(DMR) -AGH cell-derived SC mice.

Fig. S1E shows expression analysis of imprinted gene (Gtl2 and Dlk1) invarious organs of H19^(Δ) ^(DMR) -AGH cell-derived normal andgrowth-arrested SC mice.

Fig. S1F shows severely erased methylation imprinting of IG-DMR ingrowth-arrested SC mice.

Fig. S1G shows Cobra assay of IG-DMR in growth-arrested and normal SCmice.

Fig. S1H shows methylation state of IG-DMR in H19^(Δ) ^(DMR) -AGH cellline.

FIG. 2A shows semi-cloned mice produced with IG^(Δ) ^(DMR) -AGH-1 cellline (P17), in which asterisk represents growth-arrested SC mice, whichdie shortly after birth.

FIG. 2B shows methylation state of H19 DMR in normal and abnormal SCmice produced with IG^(Δ) ^(DMR) -AGR-1 cell line.

FIG. 2C schematically shows H19 DMR knockout sgRNAs designed.

FIG. 2D shows genotyping of H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGH-OG3 celllines, produced by knocking out H19 and IG-DMRs from AGH-OG3 cell line.

FIG. 2E shows genotyping of offspring obtained by mating SC miceproduced with DKO-AG-haESCs, where the offspring carrying IG-DMR diebefore or shortly after birth.

FIG. 2F shows analysis of gene expression profile of DKO-AG-haESCs byRNA-seq. The gene expression profile is clustering of the expression ofall genes. The three DKO-AG-haESC cell lines show a gene expressionprofile that is similar to that of the AG-haESCs in the control group,but very different from that of the round spermatid. To avoid the effectof haploid diploidization on the expression profile, G0/G1 cells arecollected by FACS for RNA-seq.

FIG. 2G shows imprinted gene expression profile of DKO-AG-haESCs. Theexpression profiles of three different DKO-AG-haESC lines are similar tothat of the AG-haESCs in the control group, but different from that ofthe round spermatids in mice

FIG. 2H shows analysis of methylation in DKO-AG-haESCs by RRBS.

Fig. S2A shows genotyping of H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGH cellline obtained by knocking out IG-DMR from H19^(Δ) ^(DMR) -AGH cells.

Fig. S2B shows the sequencing result of a DKO-AG-haESC cell line(H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGH-4).

Fig. S2C shows 2-cell embryos obtained with H19^(Δ) ^(DMR) -IG^(Δ)^(DMR) -AGR cells by ICAHCI.

Fig. S2D shows genotyping of SC mice, where H19^(Δ) ^(DMR) -IG^(Δ)^(DMR) -AGH-4-derived SC mice all have H19 and IG DMR knockouts.

Fig. S2E shows SC mice produced with IG^(Δ) ^(DMR) -AGH-2 (p8).

Fig. S2F shows the methylation state of H19 DMR in IG^(Δ) ^(DMR) -AGHcells.

Fig. S2G shows the methylation state of H19 DM in IG^(Δ) ^(DMR)-AGH-2-derived normal and growth-arrested SC mice.

Fig. S2H shows sequencing of a DKO-AG-haESC cell line (IG^(Δ) ^(DMR)-H19^(Δ) ^(DMR) -AGH-2).

Fig. S2I shows SC mice produced with IG^(Δ) ^(DMR) H19^(Δ) ^(DMR) -AGH-2cells (p24) by ICAHCI.

FIG. 52J shows genotyping of IG^(Δ) ^(DMR) -H19^(Δ) ^(DMR)-AGR-2-derived SC mice.

FIG. 3A is a view schematically showing Tet1, Tet2 and Tet3 sgRNAs.

FIG. 3B shows mCherry positive cells collected by FACS, which are platedin a Petri dish, to obtain DKO-AG-haESCs with mutant Tet family ofgenes.

FIG. 3C shows a Tet-TKO-DAH cell line obtained.

FIG. 3D shows sequencing of Tet1, Tet2 and Tet3 in the Tet-TKO-DAH cellline.

FIG. 3E shows sequencing of PCR products of Tet1, Tet2 and Tet3 genes inSC mice produced with Tet-TKO-DAH-1 and Tet-TKO-DAH-2 cell lines.

FIG. 3F is a view schematically showing p53, p63 and p73 sgRNAs.

FIG. 3G shows sequencing of p53, p63 and p73 in p53-TKO-DAH-2 cell line.

FIG. 3H is a view schematically showing Tet1-EGFP, Tet2-mCherry, andTet1-ECFP double-stranded DNA vectors, in which EGFP, mCherry, and ECFPare respectively fused to a stop codon of Tet1, Tet2, and Tet3.

FIG. 3I shows genotyping of Tet-TKO-DAH-1 cell line.

Fig. S3A shows sequencing of 2 DKO-AG-haESCs cell lines (H19^(Δ) ^(DMR)-IG^(Δ) ^(DMR) -AGH-OG3-1 and H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGH-OG3-1)obtained by knocking out H19 and IG-DMR from WT-AG-haESCs (AGH-OG3).

Fig. S3B shows SC mice produced with H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR)-AGU-OG3-1 (p26) by ICAHCI.

Fig. S3C shows genotyping of SC mice produced with H19^(Δ) ^(DMR)-IG^(Δ) ^(DMR) -AGH-OG3-2 cell line.

Fig. S3D shows expression analysis of imprinted gene (H19, Igf2, Gtl2and Dlk1) in DKO-AG-haESCs and normal AG-haESCs, where after H19 andIG-DMR are knocked out, the expression of H19 and Gtl2 in AGH-OG-3 isdown regulated, while the expression of Igf2 and Dlk1 is up regulated.

Fig. S3E shows bigwig track of H19 and Gtl2. The RNA-seq result showsthat compared with WT-AG-haESCs, the expression level of H19 and Gtl2 inDKO-AG-haESCs is much lower.

Fig. S3F shows the methylation levels in H19-Igf2 and Dlk1-Dio3imprinted cluster regions.

Fig. S3G shows the methylation state of imprinted genes inDKO-AG-haESCs, WT-AG-haESCs, and round spermatids.

FIG. 4A is a view schematically showing a large number of heterozygousmutant SC mice produced with DKO-AG-haESCs carrying sgRNA library byICAHCI.

FIG. 4B represents mCherry-positive haploid cells successfullytransfected with Cas9 enriched by FACS for subsequent ICAHCI.

FIG. 4C shows PCR identification of sgRNAs in single cell derivedhaploid clones, where the tested cell clones all carry sgRNAs.

FIG. 4D shows sequencing of different mutant genes in cell clones, wherethe tested cell clones all have modifications of the genes of interest.

FIG. 4E shows SC mice derived from DKO-AG-haESCs carrying sgRNA library.

FIG. 4F shows PCR identification of sgRNAs in SC mice.

FIG. 4G shows sequencing of different mutant genes of interest in SCmice, where all the tested SC mice have modifications of the genes ofinterest.

Fig. S4A shows sequencing of Tet1, Tet2 and Tet3 in Tet-TKO-DAH-3 cellline.

Fig. S4B shows SC mice produced with Tet-TKO-DAH-3 (p36) by ICAHCI.

Fig. S4C shows sequencing of p53, p63 and p73 in p53-TKO-DAH-1 cellline.

Fig. S4D shows SC mice produced with p53-TKO-DAH-1 cell line (p44) byICAHCI.

Fig. S4E shows sequencing of p53, p63 and p73 in SC mice produced withp53-TKO-DAH-1 and p53-TKO-DAH-2 cell line.

FIG. 5A is schematic representation of a large number of bi-allelicmutant SC mice produced by ICAHCI with DKO-AG-haESCs that carryconsistently expressed Cas9 and sgRNA library.

FIG. 5B shows PCR analysis of Cas9 in single cell-derived cell clone,where all tested cell clones comprises the Cas9 transgene.

FIG. 5C shows quantitative PCR analysis of Cas9 in single cell-derivedcell clones.

FIG. 5D shows PCR identification of sgRNAs in single cell-derived cellclones.

FIG. 5E shows SC mice derived from DKO-AG-haESCs that carry constantlyexpressed Cas9 and sgRNA library.

FIG. 5F shows PCR identification of sgRNAs in SC mice.

FIG. 5G shows bi-allelic mutant SC mice produced by ICAHCI withDKO-AG-haESCs that carry constantly expressed Cas9 and sgRNA Library, inwhich the Polm bi-allelic mutant SC mice are taken as an example.

FIG. 5H shows analysis of the polm gene mutation in the mouse tail by TAcloning and sequencing, where 24 out of 26 tested clones have aframeshift mutation.

FIG. 5I shows summary of TA cloning and sequencing results of 7bi-allelic mutant SC mice, in which more than 80% of the clones haveinsertion or deletion mutations.

FIG. 5J shows TA cloning and sequencing of different organs in Scube1bi-allelic mutant SC mice.

Fig. S5A is a view schematically showing Tet1-EGFP, Tet2-mCherry, andTet3-ECFP exogenous double-stranded vectors.

Fig. S5B shows genotyping of Tet1-EGFP knock-in DKO-AG-haESCs.

Fig. S5C shows genotyping of Tet3-ECFP knock-in DKO-AG-haESCs.

Fig. S5D shows genotyping of Tet1&3-KI-DAH-1 cell line.

Fig. S5E shows sequencing of Tet1-EGFP and Tet-ECFP in Tet1&3-KI-DAH-1cell line.

Fig. S5F shows 3-week-old SC mice produced with Tet1&3-KI-DAH-1 cellline (p40) by ICAHCI.

Fig. S5G shows genotyping of SC mice produced with Tet1&3-KI-DAH-1 cellline.

Fig. S5H shows sequencing of Tet2-mCherry in Tet-TKI-DAH-1 cell line.

Fig. S5I shows SC mice produced with Tet-TKI-DAH-2 cell line (p50).

Fig. S5J shows genotyping of SC mice produced with Tet-TKI-DAH-1 cellline.

Fig. S6A shows bi-allelic mutant mice produced by injecting haploidcells transiently treansfected with pX330-mCheny plasmid and carryingsgRNAs into mature oocytes and then injecting Cas9 mRNA to thereconstructed oocytes (where the protocol is known asLenti-sgRNA+pX330+Cas9 injection).

Fig. S6B shows PCR identification of sgRNA insertion into singlecell-derived haploid ES clones.

Fig. S6C shows SC mice produced with DKO-AG-haESCs carrying sgRNAlibrary.

Fig. S6D shows PCR identification of sgRNAs in SC mice.

Fig. S6E shows bi-allelic mutant mice produced by injectingDKO-AG-haESCs carrying sgRNA library into oocytes, and then injectingCas9 mRNA to SC embryos, in which bi-allelic mutant mice carryingSlco5al gene are taken as an example.

Fig. S6F shows TA cloning and sequencing of tails of mice carryingSlco5al gene, where 18 out of the 20 tested clones have insertion ordeletion mutations.

Fig. S6G shows summary of TA cloning and sequencing results of 4bi-allelic mutant mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides an androgenetic haploid embryonic stemcell (AG-haESC), in which H19DMR and IG-DMR are knocked out.

The AG-haESCs have a whole genome derived from spermatid, has theself-replication ability and pluripotency of stem cells, and can replacethe spermatid to combine with oocytes to support the full development ofembryos.

H19 DMR refers to a differentially methylated region (DMR) within theH19-Igf2 imprinted cluster. The specific location and sequence of H19DMR can be determined according to the existing methods such asmethylation sequencing or homologous sequence analysis and prediction.Human H19DMR is known to be located in the 1p15.5 region of thechromosome 1 and the mouse H19 DMR is located at the distal end ofchromosome 7 between the two genes H19 and Igf2, a position from 2 kb to4 kb upstream of the H19 gene. H19 DMR is methylated on the paternalallele, resulting in the inability of CTCF protein to bind to thismethylated region so that the enhancer downstream of H19 does not needto cross over the obstacle CTCF, thereby increasing the expression ofupstream Igf2 and decreasing the H19 expression. H19 DMR is demethylatedon the maternal allele, and the CTCF protein is able to bind to thisunmethylated region, so the enhancer downstream of H19 can only increasethe H19 expression, and cannot regulate the upstream Igf2. If thepaternal H19 DMR is knocked out, the enhancer downstream of H19 canupregulate the expression of Igf2. Since the androgenetic haploid is ofpaternal origin, theoretically it should be in a completely methylatedstate. However, studies show that the H19 DMR in the androgenetichaploid cultured in vitro suffers from abnormally erased methylation,and becomes demethylated, so that the expression of H19 is abnormallyup-regulated and the expression of Igf2 is down-regulated. In thepresent invention, H19 DMR is knocked out and the abnormal state inwhich H19 expression is up-regulated and the Igf2 expression isdown-regulated is corrected.

IG-DMR refers to a differentially methylated region (DMR) within theDlk-Dio3 imprinted cluster. The specific location and sequence of IG DMRcan be determined according to the existing methods such as methylationsequencing or homologous sequence analysis and prediction. The mouseIG-DMR is known to be located on the chromosome 12 in a 4.15 kb repeatbetween the genes Dlk1 and Gtl2 in the imprinted cluster, and the humanIG-DMR is located on the chromosome 14 (14q32.2). IG-DMR is DNAmethylated on the paternal allele, so the gene Gtl2 and some micromRNAsin this imprinted cluster are not expressed while the gene Rtl1, Dlk1and Dio3 are expressed. IG-DMR is un-DNA methylated (in demethylatedstate) on the maternal allele, so Gtl2 and some micromRNAs are expressedwhile the gene Rtl1, Dlk1 and Dio3 are not expressed. In theandrogenetic haploid (of paternal origin) and SC animals born abnormal,studies show that the normally methylated IG-DMR suffers from abnormallyerased methylation, causing the silencing of the genes Rtl1, Dlk1, andDio3, and the abnormal activation of Gtl2 and some microRNAs.

Further, the AG-haESCs undergo other genetic modifications in additionto H19 DMR and IG-DMR knockouts.

Specifically, genetic modification refers to the structural change of agene made by a biological, chemical or physical means compared with thatbefore modification, and this change mainly refers to the change in basepair composition, comprising, but not limited to, changes caused by thereplacement, insertion, and deletion of one or more base pairs.

In a preferred embodiment, the genetic modifications of Tet1, Tet2, Tet3and p53 family of genes are exemplified.

The AG-haESCs are derived from mammals, comprising human or non-humanmammals. Preferably, the AG-haESCs are derived from a rodent, such asrabbit and murine that may be a mouse or a rat. In a preferredembodiment, the AG-haESCs are derived from mice.

Compared with AG-haESCs in which H19 DMR and IG-DMR are not both knockedout, the birth rate of semi-cloned animals constructed with theAG-haESCs of the present invention is higher.

The present invention also provides a method for preparing theAG-haESCs, which comprises knocking out the H19 DMR and IG-DMR fromAG-haESCs, to obtain the AG-haESCs.

The H19 DMR and IG-DMR can be knocked out by using an existing geneediting method. In a preferred embodiment, the H19 DMR and IG-DMR areknocked out using CRISPR/Cas9-mediated gene manipulation. Gene knockoutsmay also be performed by other methods, and the present invention is notlimited to the methods listed in the examples.

Due to the H19 DMR knockout, the complete sequence of H19 DMR is removedfrom the chromosome DNA; and due to the IG DMR knockout, the completesequence of IG-DMR is removed from the chromosome DNA.

In an embodiment, H19 DMR knockout AG-haESCs are constructed firstly,and then IG-DMR is further knocked out. In another embodiment, IG-DMRknockout AG-haESCs are constructed firstly and then H19 DMR is furtherknocked out. In another example, AG-haESCs in which H19 DMR and IG-DMRare both knocked out are directly constructed.

Further, the AG-haESCs also undergo other genetic modifications.

Such other genetic modifications refer to genetic modifications otherthan H19 DMR and IG-DMR knockouts. Such other genetic modifications maybe the modification of a single target gene or the modifications ofmultiple target genes of interest. The target gene of interest is notspecific and can be set and modified as desired in the research. Forexample, such other genetic modifications may be the modifications ofone, two or more target genes. The AG-haESCs of the present invention inwhich H19 DMR and IG-DMR are both knocked out can be passaged in vitro,and thus they can theoretically be genetically modified constantly. Thenumber of modifications made to the target gene can be set as neededwithout particular limitation.

The genetic modifications comprise, but are not limited to, knock-in andknock-out of a target gene, and the like. The knock-in and knock-out ofa target gene may be accomplished by techniques such as gene targetingand homologous recombination, comprising, but not limited to, geneticmanipulation based on ZFN (zinc finger nuclease), TALEN (transcriptionalactivator-like effector nuclease) and CRISPR/Cas9 (clustered regularlyinterspaced short palindromic repeats).

In an embodiment, the AG-haESCs in which the H19 DMR and IG-DMR are bothknocked out undergo one or more genetic modifications to obtain theAG-haESCs with H19 DMR and IG-DMR knockouts and with other geneticmodifications. Alternatively, AG-haESCs can be genetically modifiedfirst, followed by knocking out H19DMR and IG-DMR from the geneticallymutated AG-haESCs. In addition to the above, other biotechnologicalmeans that can achieve the H19 DMR and IG-DMR knockouts and othergenetic mutations can also be used to construct the AG-haESCs with H19DMR and IG-DMR knockouts and with other genetic modifications.

The present invention also provides the use of the AG-haESCs inconstructing genetically modified semi-cloned animals. Further, theAG-haESCs are used as a fertilizing vector in place of spermatid in theconstruction of genetically modified animals.

The present invention also provides a method for constructing agenetically modified semi-cloned animal, which comprises: combining anAG-haESC in which H19 DMR and IG-DMR are both knocked out with an oocyteto obtain a semi-cloned embryo, and incubating the semi-cloned embryo toobtain a semi-clone animal.

In general, the oocytes and the AG-haESCs are derived from the same kindof animal, preferably the same species of animal.

The semi-cloned embryo may specifically be an semi-cloned embryoobtained by ICAHCI using the AG-haESCs in which H19 DMR and IG-DMR areboth knocked out as a donor for ICAHCI.

Further, the semi-cloned animal can be obtained by incubating thesemi-cloned embryo in a suitable female organism by embryo transfer. Ina preferred embodiment, the suitable maternal is a pseudopregnant ICRfemale rat.

Further, the AG-haESCs undergoes other genetic modifications.

The present invention further provides a genetically modified animal,which is constructed according to the above method, or is a sexuallyreproduced offspring of a semi-cloned animal constructed according tothe above method.

The semi-cloned animal according to the present invention may be anon-human mammal. Preferably, the semi-cloned animal is a rodent, suchas rabbit or murine. In a preferred embodiment, the semi-cloned animalis a mouse.

The present invention also provides a method for constructing agenetically modified semi-cloned animal library, which comprises thesteps of:

1) infecting the AG-haESCs according to the present invention with virusparticles prepared with the lentiviral sgRNA library plasmids, to obtainan AG-haESC library carrying the sgRNA library;

2) obtaining a semi-cloned embryo library by ICAHCI using a vectorexpressing Cas9 and/or Cas9 mRNA, with AG-haESCs in the AG-haESC librarycarrying the sgRNA library as a donor for ICAHCI; and

3) incubating the embryos in the semi-cloned embryo library to obtain asemi-cloned animal library.

The sgRNA lentiviral library plasmids comprise several lentiviralvectors that express different sgRNAs. The lentiviral sgRNA library canbe constructed by current technologies, or existing lentiviral sgRNAlibrary plasmids may be used. Specifically, sgRNAs designed fordifferent genes can be cloned into lentiviral vectors. The sgRNA can bedesigned according to the gene of interest.

In a preferred embodiment, a commercially available lentiviral sgRNAlibrary of whole genomes of mice is employed.

Specifically, Step 2) may be selected from any one of:

Method A:

The AG-haESC library carrying the sgRNA library is further transfectedwith a plasmid expressing Cas9, and the semi-cloned embryos are obtainedby ICAHCI using the resultant AG-haESCs as a donor for ICAHCI.

Method B:

AG-haESCs in the AG-haESC library carrying the sgRNA library, as a donorfor ICAHCI, are injected into mature oocytes by ICAHCI, and then Cas9mRNA is injected into the reconstructed oocytes, to obtain thesemi-cloned embryos.

Method C:

The AG-haESC library carrying the sgRNA library is further transfectedwith a plasmid expressing Cas9, the resultant AG-haESCs, as a donor forICAHCI, are injected into mature oocytes by ICAHCI, and then Cas9 mRNAis injected into the reconstructed oocytes, to obtain the semi-clonedembryo, from which a semi-cloned animal is obtained after embryotransfer.

In the methods A and C, the plasmid expressing Cas9 can be constructedby cloning the Cas9 expressing gene into an expression plasmid. In apreferred embodiment, the plasmid expressing Cas9 is pX330-mCherryplasmid. The plasmid that is constructed to express Cas9 is not limitedto the pX330 plasmid. The expression plasmid only needs to be suitablefor expression of exogenous genes in mammalian cells.

The present invention further provides another method for constructing agenetically modified semi-cloned animal library, which comprises thesteps of:

1) infecting the AG-haESCs according to the present invention withlentiviral particles expressing Cas9 and lentiviral particles preparedwith the lentiviral sgRNA library, to obtain an AG-haESC library withconstant expression of the sgRNA library and Cas9;

2) obtaining a semi-cloned embryo library by ICAHCI with AG-haESCs inthe AG-haESC library with constant expression of the sgRNA library andCas9 as a donor for ICAHCI; and

3) incubating the embryos in the semi-cloned embryo library to obtain asemi-cloned animal library.

The virus particles expressing Cas9 can be obtained by cloning theencoding gene expressing Cas9 into a lentiviral vector and thenpackaging the lentivirus in the prior art. The lentiviral vectorexpressing Cas9 is commercially available.

The semi-cloned animal library comprises several genetically mutatedsemi-cloned animals. The animals may be heterozygous or biallelic mutantanimals.

Further, semi-cloned animals can be obtained by culturing thesemi-cloned embryos in a suitable female organism by embryo transfer. Ina preferred embodiment, the suitable female organism may be apseudopregnant ICR female rat.

The present invention also provides a genetically modified semi-clonedanimal library, which is constructed according to the method asdescribed above.

The genetically modified semi-cloned animal library of the presentinvention can be used in genetic screening of genes at subordinateindividual level.

The semi-cloned animal library of the present invention may be anon-human mammalian library. Preferably, the semi-cloned animal libraryis a rodent library, such as a rabbit library, and a murine library. Ina preferred embodiment, the semi-cloned animal library is a mouselibrary.

The embodiments of the present invention are described below withreference to specific examples, and other advantages and effects of thepresent invention can be easily understood by those skilled in the artfrom the disclosure of the present invention. The present invention canalso be implemented or practiced through additional different specificembodiments. The details in this specification may also be based ondifferent perspectives and applications, and various modifications orchanges can be made without departing from the spirit of the presentinvention.

When a numerical range is given in an example, it is to be understoodthat both endpoints of each numerical range and any numerical valuebetween the two endpoints are encompassed, unless the context otherwiseindicates. Unless defined otherwise, all technical and scientific termsas used herein have the same meanings as those commonly understood bythose skilled in the art. In addition to the specific methods, equipmentand materials used in the examples, the present invention may beimplemented using any of the methods, devices, and materials in theprior art that are similar or equivalent to the methods, devices, andmaterials described in the examples of the present invention, based onthe knowledge of those skilled in the art the prior art and thedisclosure of the present invention,

Unless otherwise specified, in the experimental methods, detectionmethods, and preparation methods disclosed in the present invention, theconventional molecular biology, biochemistry, chromatin structure andanalysis, analytical chemistry, cell culture, recombinant DNA technologyand conventional techniques in related fields are adopted. Thesetechniques are well documented in the literature.

Abbreviations

AG-haESCs: androgenetic haploid embryonic stem cells

DKO-AG-haESCs: H19 DMR and IG-DMR double knockout androgenetic haploidembryonic stem cell

1. Experimental Materials and Methods

1.1. Materials and Reagents

The cell culture medium (DMEM), fetal bovine serum (FBS), serumreplacement (KSR), trypsin, Opti-MEM, DPBS, and Lipofectamine 2000 werepurchased from Life Technologies Inc.; the restriction endonucleases andT4 ligase were purchased from NEB; the Taq enzyme and dNTPs werepurchased from TaKaRa; the CDNA reverse transcription kit, andfluorescent quantification reagent SYBR-Green were purchased fromTOYOBO; and the oligonucleotides were synthesized by Shanghai GenerayCompany.

HEPES-CZB culture medium

H-CZB Stock 98.5 ml, Hepes.2Na (sigma, CAT#H0763) or ICN 520 mg or Hepes(sigma, CAT#H4034) 476 mg, NHCO₃ 42 mg, CaCl₂.2H₂O 100× stock 1 ml,Pyruvate 3.0 mg, and Glutamin 200× stock 0.5 ml, adjusted to pH 7.4, andfiltered well.

H-CZB stock:

CZB stock 500 ml, PVA (sigma, P8136) 50 mg, CZB stock: H₂O 985 ml, NaCl(sigma, CAT#55886) 4760 mg, KCL (sigma, CAT#P5405) 360 mg, MgSO₄.7H₂O(sigma, CAT#M1880) 290 mg, EDTA-2Na (sigma, CAT#E6635) 40 mg, Na-Lactate(sigma, CAT#L7900) 5.3 ml, D-Glucose (sigma, CAT#G6152) 1000 mg, andKH₂PO₄ (sigma, CAT#P5655) 160 mg

Activation solution: 10 mM Sr2+, 5 ng/ml Trichostatin A (TSA)

KSOM culture medium (KSOM+AA with glucose): millipore, CAT#MR-106-D

ESC medium:

DMEM (millipore, CAT#SLM-220-M) 75%, 20% serum replacement KSR (Gibco,CAT#10828-028), 1,500 U/ml LIF (Millopre, CAT#ESG1107), 3M CHIR99021(Stemgent, CAT#04-0004), and 1M PD0325901 (Stemgent, CAT#04-0006)

Acid Tyrode solution: sigma, CAT#T1788

CZB culture medium:

CZB stock 99ML, CaCl₂.2H₂O 100× stock 1 ml, Pyruvate 3.0 mg, Glutamin200× stock 0.5 ml, BSA 500 mg

pX330-mCherry:

enzymatically cleaving px330 (addgene) plasmid with Notl, and theninserting a CMV-mcherry-pA fragment amplified from pmCherry-C1(Clontech) into the enzymatically cleaved px330 plasmid.

Primers for amplification: mCherry-F: ATTTGCGGCCGCATAGTAATCAATTACGGGmCherry-R: ATTTGCGGCCGCATGCAGTGAAAAAAATGC

Lentiviral sgRNA library of mice: supplied by Addgene

viral plasmid expressing Cas9: supplied by Addgene

Cas9 mRNA:

obtained as described in Wang, H., Yang, H., Shivalila, C. S., Dawlaty,M. M., Cheng, A. W., Zhang, F., and Jaenisch, R. (2013). One-stepgeneration of mice carrying mutations in multiple genes byCRISPR/Cas-mediated genome engineering. Cell 153, 910-918.

Round spermatid:

The mouse testis was digested with collagenase IV for 20 minutes andthen with trypsin for 10 minutes, and then sorted by FACS, to obtainround spermatid of mice.

1.2. Test Animals

All animals were used in accordance with the procedures in the animaloperation manual of Institute of Biochemistry and Cells, ShanghaiInstitutes for Biological Sciences, Chinese Academy of Sciences.

H19 Δ3.8 kb KO mice (C57/B6 background, homozygous): constructed asdescribed in (Thorvaldsen, J. L., Mann, M R., Nwoko, O., Duran, K. L.,and Bartolomei, M. S. (2002). Analysis of sequence upstream of theendogenous H19 gene reveals elements both essential and dispensable forimprinting. Molecular and cellular biology 22, 2450-2462.)

IG-DMRKO mice (C57/B6 background, heterozygous): constructed asdescribed in (Lin, S. P., Youngson, N., Takada, S., Seitz, H., Reik, W.,Paulsen, M., Cavaille, J., and Ferguson-Smith, A C. (2003). Asymmetricregulation of imprinting on the maternal and paternal chromosomes at theDlk1-Gtl2 imprinted cluster on mouse chromosome 12. Nature genetics 35,97-102)

B6D2F1 (C57BL/6×DBA2) female mice: female offspring obtained aftermating with female mice of C57BL/6 strain with male mice of DBA2 strain.

Pseudopregnant ICR female mice: ICR adult female mice purchased fromSLAC Laboratory Animal Co., Ltd were mated with ligated adult ICR malemice, to obtain pseudopregnant ICR female rats.

1.3. Establishment of AG-haESC Line

The AG-haESC line was constructed according a reported method (Yang, H.,Shi, L., Wang, B. A., Liang, D., Zhong, C., Liu, W., Nie, Y., Liu, J.,Zhao, J., Gao, X., et al. (2012). Generation of genetically modifiedmice by oocyte injection of androgenetic haploid embryonic stem cells.Cell 149, 605-617).

Method:

The MII oocytes were enucleated, into which the corresponding spermheads were injected. Mouse MII oocytes were harvested 14 hours aftertreatment with human chorionic gonadotropin (HCG) and then enucleatedusing a Piezo needle in HEPES-CZB medium containing 5 μg/ml cytochalasinB (CB). After enucleation, single sperm heads were injected into thecytoplasm of the oocytes. The reconstructed embryos were cultured in CZBmedium for 1 hour and then transferred to the activation solutioncontaining 1 mM Sr²⁺ for activation. After activation, all reconstructedembryos were transferred to KSOM medium containing amino acids andincubated at 37° C., and 5% CO₂. The reconstructed embryos reaching themorula or blastula stage 3.5 days later were seeded in ESC medium.

The zona pellucid of the reconstructed embryos was removed by digestionwith the acid Tyrode solution. Each embryo was transferred to a 96-wellplate plated with a mouse fibroblast feeder layer and cultured with ESCmedium containing 20% serum replacement (KSR), 1,500 U/ml LIF, 3MCHIR99021 and 1 M PD0325901. After 4-5 days of culture, the cell cloneswere trypsinized and transferred to a 96-well plate plated with a freshfeeder layer. The cells were further expanded, and passaged into a48-well plate and further into a 6-well plate, and the cells were dailymaintained in a 6-well plate. To sort the haploid cells, after theembryonic stem cells were trypsinized, they were washed once with PBS(GIBCO) and then in ESC medium containing 15 μg/ml Hoechst 33342. Afterbeing placed in a water bath for 30 min, haploid cells of 1N peak weresorted by the flow cytometer BD FACS Ariall and subsequently subculturedto obtain the AG-haESC line.

1.4 CRISPR-Cas9-Mediated Genetic Manipulation

Construction of CRISPR-Cas9 plasmid: The synthesized forwardoligonucleotide strand and reverse oligonucleotide strand of sgRNA wereannealed to obtain a double-stranded oligonucleotide strand (in thepresent invention, the sgRNA sequence refers to the sequence of theforward oligonucleotide strand of the sgRNA), which was then ligated topX330-mCherry enzymatically cleaved with BbsI (New England Biolabs). Theconstructed corresponding plasmid was transfected into the AG-haESCsusing Lipofectamine 2000 (Life Technologies) according to theinstructions. 48 hours after transfection, the haploid cells with thered fluorescent protein were sorted by flow cytometry (FACSAriaII, BDBiosciences) and then plated at a low density. After 4-5 days of growth,monoclones were picked up for subsequent construction of cell lineages.Finally, cell lines with corresponding gene mutations were obtained bysequencing target genes by PCR.

If gene knock-in was involved, a double-stranded DNA donor needed to beconstructed.

Preparation of double-stranded DNA donor:

A sequence encoding EGFP, mCherry or ECFP was amplified and then ligatedto the pMD19-T vector, to give pMD19-T-EGFP/mCherry/ECFP. Subsequently,the left and right homologous arms of the target gene were inserted intothe pMD 19-T-EGFP/mCherry/ECFP vector.

1.5. Virus Production and Viral Infection of Double Knockout AG-haESCs(DKO-AG-haESCs)

The viral sgRNA library of mice and the Cas9 expressing viral plasmidhave been reported mice (Cong., L, Ran, F. A., Cox, D., Lin, S.,Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, LA., et al. (2013). Multiplex genome engineering using CRISPR/Cassystems. Science 339, 819-823; and Koike-Yusa, H., Li, Y., Tan, E. P.,Velasco-Herrera Mdel, C., and Yusa, K. (2014). Genome-wide recessivegenetic screening in mammalian cells with a lentiviral CRISPR-guide RNAlibrary. Nature biotechnology 32, 267-273, 2014). The viral sgRNAlibrary of mice and the Cas9 expressing viral plasmid used in thepresent invention were provided by Addgene. To prepare the virus,HEK293T was passaged in advance into a 10 cm petri dish, 3 μg of viralplasmid (lentiviral sgRNA library or lentiviral Cas9) and 9 μg ofViraPower Lentiviral Packaging Mix (Invitrogen) were transfected intoHEK293T cells by using Lipofectamine® 2000 Reagent (Invitrogen, LifeTechnologies). The supernatant were collected 72 hours aftertransfection and concentrated with Lenti-Concentin virus precipitationsolution (SBI) and then stored at −80° C.

Infection with lentiviral Cas9: A cell suspension of 10⁸ DKO-AG-haESCswas infected for 48 hours with 8 μg/ml polybrene (Sigma) and packagedlentiviral Cas9, and then screened for 3 days with 10 μg/ml blasticidin(Sigma). The remaining resistant clone was a cell line integrated withlentiviral Cas9.

Infection with lentiviral sgRNA library: A cell suspension of 10⁸DKO-AG-haESCs was infected for 48 hrs with 8 μg/ml polybrene (Sigma) andpackaged lentiviral CRISPR-sgRNA library, and then screened for 2 daysin a medium comprising 1 μg/ml puromycin (Invitrogen), to obtain apositive clone that is a cell line carrying lentiviral sgRNA library.

Co-infection with lentiviral Cas9 and lentiviral sgRNA library:

A cell line integrated with lentiviral Cas9 was prepared first, thenfurther infected with the lentiviral sgRNA library, and then screenedfor 2 days in a medium comprising 1 μg/ml puromycin (Invitrogen), toobtain a positive clone that is a cell line integrated with lentiviralCas9 and lentiviral sgRNA library.

If the DKO-AG-haESC cell line was only infected with the lentiviralsgRNA library, then the cells did not express Cas9. At this time,transfection with the pX330-mCherry plasmid at the cellular level wasneeded to achieve the genome editing.

1.6. Bisulfite Sequencing for Methylation

1) Mouse DNA was packaged into beads with 15 μl of 2% LMP agarose (lowmelting point agarose), 460 μl of DNA digestion buffer, and then 40 μlof proteinase K were added to each sample and the sample was digested byincubation overnight at 50° C.

2) After 3 washes with TE, the beads were reacted with a bisulfatesolution and incubated at 50° C. for 4-8 hours.

3) Nested PCR was performed using the beads as a template, and the PCRproduct was recovered and then ligated to the PMD19-T vector, followedby transformation and plating.

4) 10 colonies were picked from each sample for sequencing.

If the EZ DNA methylation Gold kit (ZYMO Research) was used, anappropriate amount of DNA was prepared, and the following procedureswere operated according to the steps of use of the kit. The resultingproduct recovered with the kit was used as a template for PCR, theproduct was recovered and then ligated to the PMD19-T vector, followedby transformation and plating. 10 colonies were picked from the platefor sequencing.

1.7. Fluorescent Quantitative PCR

Total RNA was extracted from the cells or organs with Trizol reagent(Invitrogen) and then 1 μg of total RNA was reversely transcripted intocDNA using the First Strand cDNA Synthesis kit (TOYOBO). Real-timefluorescent quantitative PCR reactions were performed on a Bio-Rad CFX96instrument using SYBR Green Realtime PCR Master Mix (TOYOBO), with 3replicates for each set of samples. All the gene expression levels weredetected with the expression level of housekeeping gene Gapdh as aninternal reference.

1.8. Cobra Assay

1) 100 ng of sample DNA was taken and enzymatically cleaved with TaqIrestriction endonuclease (Fementas) (T/CGA) for 15 min

-   -   2) Agarose gel electrophoresis was performed.

1.9. Construction of Semi-Cloned Mice by ICAHCI, ROSI and EmbryoTransfer

Intracytoplasmic AG-haESCs injection (ICAHCI):

To obtain semi-cloned (SC) embryos, AG-haESCs were treated for 8 hrswith a medium containing 0.05 μg/ml colchicine to synchronize the cellsto M phase and then intracytoplasmically injected into the oocytes. Thedigested AG-haESCs were washed 3 times with HEPES-CZB medium and thenre-suspended in 3% (w/v) polyvinylpyrrolidone (PVP) in HEPES-CZB medium.Nuclei of AG-haESCs in M phase were injected into MII oocytes under aPiezo microscope. The reconstructed embryos were cultured in CZB mediumfor 1 hour and then activated with a CB-free medium for 5-6 hours. Afteractivation, all the reconstructed embryos were cultured in KSOM mediumat 37° C., and 5% CO₂. ICAHCI embryos reached 2-cell embryo after beingcultured in the KSOM medium for 24 hours.

ROSI (round spermatid injection):

The operation followed a reported method (Kishigami, S., Wakayama, S.,Nguyen, V. T., and Wakayama, T. (2004). Similar time restriction forintracytoplasmic sperm injection and round spermatid injection intoactivated oocytes for efficient offspring production. Biology ofreproduction 70, 1863-1869).

Every 15-20 2-cell embryos obtained by ICAHCI or ROSI were transferredinto each uterus of pseudopregnant ICR mice at 0.5 dpc (0.5 daypost-mating). The female mice experienced caesarean section or naturalbirth after 19.5 days of pregnancy. Caesarean section was done forreconstructed embryos obtained with WT AG-haESCs or single DMR knockoutAG-haESCs, and expired fetuses were quickly peeled off from the female'suterus. For embryos obtained by ROSI or with DKO-AG-haECs, females after19.5 days of pregnancy experienced natural birth. After removing thefluid from the born mice, the mice were placed in an oxygen incubator,and survived mice were subsequently nourished by the surrogate females.

1.10. RNA-Seq and Gene Expression Analysis

The RNA-seq library of total RNA was established according to Illumina'sofficial TreSeq RNA Sample Prep v2 Guide. After establishment, deepsequencing was performed on the IlluminaHiSeq 2000 instrument availablefrom the Computational Biology Center of the Institute of ComputingBiology, Chinese Academy of Sciences-Max-Planck-Gesellschaft zurFörderung der Wissenschaften e.V. 6 samples, comprising 2 WT AG-haESCs,H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGH, H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR)-AGH-OG3, IG^(Δ) ^(DMR) -H19^(Δ) ^(DMR) -AGH and round spermatid weresubjected to subsequent analysis.

The algorithm for gene expression level was RPKM, specifically asdescribed in (Yang, L., Duff, M. O., Graveley, B. R., Carmichael, G. G.,and Chen, L. L. (2011). Genomewide characterization ofnon-polyadenylated RNAs. Genome biology 12, R16).

The p-value of differentially expressed genes was calculated using thewaldscore method (Yang et al., Genome boil 2011) in which abs(waldscore) was set to be >1.96 (that is, p-value<0.05), and then thedifferentially expressed genes were screened.

1.11. RRBS (Reduced Representation Bisulfite Sequencing)

The RRBS library was established according to Illumina's officialprotocol, and then sequenced on the IlluminaHiSeq 2000 instrument (Gu,H., Smith, Z. D., Bock, C., Boyle, P., Gnirke, A., and Meissner, A.(2011). Preparation of reduced representation bisulfite sequencinglibraries for genome-scale DNA methylation profiling. Nature protocols6, 468-481.). All sequencing reads were aligned with the mouse genome.

1.12. Genotyping Methods

The extracted genomic DNA was amplified by PCR using correspondingprimers, and the PCR product was further subjected to agarose gelelectrophoresis.

Example 1

Construction of Semi-Cloned Mice Based on H19 DMR Single KnockoutAG-haESC Line

A. Construction of AG-haESC Line:

Source of spermatids: H19 Δ3.8 kb KO mice (C57/B6 background,homozygous);

Source of oocytes: B6D2F1 (C57BL/6×DBA2) female mice

The haploid sperm heads of H19 Δ3.8 kb mice were injected intoenucleated oocytes (FIG. 1A) following the method as described inSection 1.3, to obtain reconstructed blastocysts, with which AG-haESCcell lines were constructed. Three haploid cell lines were establishedfrom 250 reconstructed blastocysts (which were designated as H19^(Δ)^(DMR) -AGH-1, H19^(Δ) ^(DMR) -AGU-2, H19^(Δ) ^(DMR) -AGU-3) (FIGS.1B-1D).

B. Construction of Semi-Cloned Mice:

Following the method as described in Section 1.9, H19-AGH cells wereused as a donor for ICAHCI, and 1443 2-cell embryos reconstructed withthese 3 cell lines were transferred into the pseudopregnant female mice.Finally, 86 healthy, viable semi-cloned mice and 39 growth-arrested micewere obtained by caesarean section of the female mice on day 19.5 afterpregnancy (FIG. 1E, Table 1). The birth rate in normal half-cloned miceis about 5.9%; however, about 2.7% of the semi-cloned mice developedfrom the embryos constructed with H19^(Δ) ^(DMR) -AGH still haveabnormal growth.

Semi-cloned mice were constructed with wild-type AG-haESCs as a donorfor ICAHCI. The birth rate of normal semi-cloned mice is about 0.7-1.8%.

C. Detection of Gtl2 Expression

Fluorescent quantitative PCR was used to detect whether aberrant highexpression of Gtl2 was also present in the major organs of H19^(Δ)^(DMR) -AGH-derived growth-arrested mice, following the method asdescribed in Section 1.7.

Primer sequences for fluorescent quantitative PCR: Gtl2-F:TTGCACATTTCCTGTGGGAC Gtl2-R: AAGCACCATGAGCCACTAGG

The experimental results show that Gtl2 overexpression occurs in most ofthe organs tested in growth-arrested semi-cloned mice compared to normalmice (Fig. S1E).

D. Methylation Analysis

Experimental Methods: The analysis was carried out following the methodsas described in Sections 1.6 and 1.8.

Primers used: IG DMR-BS-OF: TTAAGGTATTTTTTATTGATAAAATAATGTAGTTTIGD MR-BS-OR: CCTACTCTATAATACCCTATATAATTATACCATAA IG DMR-BS-IF:TTAGGAGTTAAGGAAAAGAAAGAAATAGTATAGT IG DMR-BS-IR:TATACACAAAAATATATCTATATAACACCATACAA

The results of methylation analysis show that the growth-arrestedsemi-cloned mice show obvious hypomethylation in the IG-DMR region ofthe differential methylation sites in the Dlk1-Gtl2 imprinted cluster(Figs. S1F-G). Interestingly, a more severe loss of IG-DMR methylationimprinting occurred in the later generation of H19^(Δ) ^(DMR) -AGH(H19^(Δ) ^(DMR) -AGU-1, p16) compared to with its earlier generation(H19^(Δ) ^(DMR) -AGH-1, p7), resulting in a significantly increased rateof growth arrested mice (Fig. S1H, Table S1). This indicates that theabnormal expression of Gtl2 may be another important factor leading tothe failure in development of the semi-cloned mice obtained with H19^(Δ)^(DMR) -AGH cells.

Example 2

Construction of Semi-Cloned Mice Based on H19 DMR and IG-DMR DoubleKnockout AG-haESC Line

A. Construction of H19 DMR and IG-DMR Double Knockout AG-haESC Line:

The construction was carried out following the method as described inSection 1.4.

2 sgRNAs were designed according to the sequence between Dlk1 and Gtl2which had a 4.15 kb IG-DMR knocked out (designated as IG-DMR-sgRNA1 andIG-DMR-sgRNA2) (FIG. 1F).

IG-DMR-sgRNA1 sequence: (SEQ ID NO: 1) CGTACAGAGCTCCATGGCACIG-DMR-sgRNA2 sequence: (SEQ ID NO: 2) CTGCTTAGAGGTACTACGCT

The plasmids pX330-mCherry expressing Cas9 and IG-DMR-sgRNAs wereconstructed and transfected into the H19^(Δ) ^(DMR) -AGH cells, tofinally obtain 71 AG-haESC lines. The sequencing of the PCR product ofthe target gene (IG-DMR deletion check-F: TGTGCAGCAGCAAAGCTAAG; IG-DMRdeletion check-R: ATACGATACGGCAACCAACG) found that the IG-DMR weresuccessfully knocked out in 58 cell lines (designated as H19^(Δ) ^(DMR)-IG^(Δ) ^(DMR) -AGH-1 through H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGH-58)(FIGS. 1G, S2A and S2B). Off-target analysis of H19^(Δ) ^(DMR) -IG^(Δ)^(DMR) -AGH-2 shows that no mutations occur to a total of 22 potentialoff-target sites (Table S2), where these sites are predicted bygenome-wide searching by using software reported previously (Hsu, P. D.,Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V.,Li, Y., Fine, E. J., Wu, X., Shalem, O., et al. (2013). DNA targetingspecificity of RNA-guided Cas9 nucleases. Nature biotechnology 31,827-832).

B. Construction of Semi-Cloned Mice:

Following the method as described in Section 1.9, H19^(Δ) ^(DMR) -IG^(Δ)^(DMR) -AGH cells were used as a donor for ICAHCI to constructsemi-cloned mice. The results show that 22.3% of the semi-cloned embryoscan well developed (FIG. 1H, Fig. S2C, Table 1, Table S1), which issimilar to the ROSI birth rate reported by our laboratory (Table 1) orother laboratories (Kishigami, S., Wakayama, S., Nguyen, V. T., andWakayama, T. (2004). Similar time restriction for intracytoplasmic sperminjection and round spermatid injection into activated oocytes forefficient offspring production. Biology of reproduction 70, 1863-1869.).Furthermore, compared with the semi-cloned mice obtained with wild typeAG-haESCs and H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGH by ICAHCI that requirecaesarean section, after the embryos reconstructed with H19^(Δ) ^(DMR)-IG^(Δ) ^(DMR) -AGH-2 is transferred to pseudopregnant female mice, thepseudopregnant female mice can carry out natural birth, and the bornsemi-cloned mice all survive healthy. The data suggests that H19^(Δ)^(DMR) -IG^(Δ) ^(DMR) -AGH cells obtain characteristics resembling around spermatid successfully by knocking out IG-DMR, from whichsemi-cloned mice can be produced efficiently.

Example 3

Construction of Semi-Cloned Mice Based on IG-DMR Single KnockoutAG-haESC Line

A. Construction of AG-haESC Line:

Source of spermatids: IG-DMR KO mice;

Source of oocytes: B6D2F1 (C57BL/6×DBA2) female mice

The haploid sperm heads of IG-DMR KO mice were injected into enucleatedoocytes following the method as described in Section 1.3, to obtainreconstructed blastocysts, with which AG-haESC cell lines wereconstructed. Among the 8 haploid cell lines established, 2 cell linescarried IG-DMR knockout (designated as IG^(Δ) ^(DMR) -AGH-1 and IG^(Δ)^(DMR) -AGH-2)

B. Construction of Semi-Cloned Mice:

Following the method as described in Section 1.9, IG^(Δ) ^(DMR) -AGHcells were used as a donor for ICAHC to construct semi-cloned mice. Itis found that the IG^(Δ) ^(DMR) -AGH cells are not donors effective inthe production of semi-cloned mice (Table 1, Table S1), with which it isdifficult to obtain healthy normal SC mice (where only 4 normal SC miceare obtained from 499 transferred embryos) (FIGS. 2A and S2E). Moreover,most of the mice are growth arrested.

C. Methylation Analysis

Experimental Methods: The analysis was carried out following the methodsas described in Sections 1.6 and 1.8.

Primers used: H19 DMR-BS-OF: 5′ GAGTATTTAGGAGGTATAAGAATT 3′H19 DMR-BS-OR: 5′ ATCAAAAACTAACATAAACCCCT 3′ H19 DMR-BS-IF: 5′GTAAGGAGATTATGTTTATTTTTGG 3′ H19 DMR-BS-IR: 5′ CCTCATTAATCCCATAACTAT 3′

The result show that the H19 DMR of IG^(Δ) ^(DMR) -AGH cell line sufferserased methylation, and complete loss of methylation occurs in thegrowth-arrested mice (FIGS. 2B, S2F, and S2G).

Example 4

Construction of Semi-Cloned Mice Based on H19 DMR and IG-DMR DoubleKnockout AG-haESC Line

A. Construction of H19 DMR and IG-DMR Double Knockout AG-haESC Line:

The construction was carried out following the method as described inSection 1.4.

H19 Δ3.8kb DMR KO sgRNA sequence: H19-3.8K sgRNA-1: (SEQ ID NO: 3)CATGAACTCAGAAGAGACTG H19-3.8K sgRNA-2: (SEQ ID NO: 4)AGGTGAGAACCACTGCTGAG

The H19 Δ3.8 kb DMR were knocked out from the IG^(DMR)-AGH cell lineobtained in the above example by the CRISPR-Cas9 method (seeThorvaldsen, J. L., Mann, M. R., Nwoko, O., Duran, K. L., andBartolomei, M. S. (2002). Analysis of sequence upstream of theendogenous H19 gene reveals elements both essential and dispensable forimprinting. Molecular and cellular biology 22, 2450-2462), and 13DKO-AG-haESC cell lines were successfully obtained (designated as IG^(Δ)^(DMR) -H19^(Δ) ^(DMR) -AGH-1 through IG^(Δ) ^(DMR) -H19^(Δ) ^(DMR)-AGH-13) (FIGS. 2C and S2H, and Table S2).

B. Construction of Semi-Cloned Mice:

Following the method as described in Section 1.9, 2 cell lines abovewere used as a donor for ICAHCI to construct semi-cloned mice. Theresults show that the 2 cell lines have the similar ability to producehealth SC mice as the H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGH cells (Figs.S2I and 2J, and Table 1, Table S1).

Example 5

Construction of Semi-Cloned Mice Based on H19-DMR and IG-DMR DoubleKnockout AG-haESC Line

A. Construction of H19-DMR and IG-DMR Double Knockout AG-haESC Line:

Initial cells: 21st-generation AGH-OG-3 cells of WT-AG-haESC cell lineAGH-OG-3

It has been reported that the 22^(nd) generation of this cell line hassubstantially lost the ability to produce healthy, semi-cloned mice(Yang, H., Shi, L., Wang, B. A., Liang, D., Zhong, C., Liu, W., Nie, Y.,Liu, J., Zhao, J., Gao, X., et al. (2012). Generation of geneticallymodified mice by oocyte injection of androgenetic haploid embryonic stemcells. Cell 149, 605-617).

The oligo of sgRNA were annealed and then the sgRNAs of H19 and IG-DMRwere respectively ligated to the BbsI digested px330-mCherry plasmid andtransformed. Plasmid was extracted from the bacterial suspensionsequenced to be correct for subsequent transfection.

The 21st generation of the AGH-OG-3 cell line was transformed with theplasmid obtained above.

12 H19 and Gtl2 DMR double knockout AG-haESC cell lines (designated asH19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) -AGH-OG3-1 through H19^(Δ) ^(DMR) -IG^(Δ)^(DMR) -AGH-OG3-12) were finally obtained (FIGS. 2D and S3A and TableS2).

B. Construction of Semi-Cloned Mice:

Following the method as described in Section 1.9, H19^(Δ) ^(DMR) -IG^(Δ)^(DMR) -AGH-OG3 cells were used as a donor for ICAHCI to constructsemi-cloned mice. Surprisingly, by injecting 2 of these cell lines intoMII oocytes, about 17% of semi-cloned embryos were able to develop fully(Fig. S3B, S3C, Table 1, and Table S1). This shows that WT-AG-haESCs,which had previously completely lost the ability to produce SC mice,were able to regain characteristics resembling a round spermatid afterknockdown of H19-DMR and IG-DMR.

Example 6

Genotyping of SC Mice Obtained with 3 DKO-AG-haESC Lines

Genotyping method: The method as described in Section 1.12 was used.

H19-DMR deletion check-F2: GTGGTTAGTTCTATATGGGGH19-DMR deletion check-R2: TCTTACAGTCTGGTCTTGGT IG-DMR deletion check-F:TGTGCAGCAGCAAAGCTAAG IG-DMR deletion check-R: ATACGATACGGCAACCAACG

Three H19-DMR and IG-DMR double-knockout AG-haESC cell lines wereconstructed by using the three methods described in the foregoingExamples 2, 4 and 5, and a total of 402 SC mice were obtained. A mousebirth rate of 20.2% was achieved with the transferred embryos. TheseDKO-AG-haESC derived SC mice are able to grow to adulthood and arecapable of producing offspring. Genotyping of 33 neonatal mouseoffspring in 7 litters find that 13 animals carry H19-DMR knockout and11 animals are WT (FIG. 2E). 6 out of the other 9 mice have IG-DMRknockout and 3 have H19 and Gtl2 DMR double knockouts. The 9 mice diedshortly after birth, consistent with the previously reported lethalphenotype of the maternally inherited IG-DMR before or after birth (Lin,S. P., Youngson, N., Takada, S., Seitz, H., Reik, W., Paulsen, M.,Cavaille, J., and Ferguson-Smith, A C. (2003). Asymmetric regulation ofimprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2imprinted cluster on mouse chromosome 12. Nature genetics 35, 97-102).

Example 7

Detection of Gene Expression and Methylation Analysis of AG-haESCs

RNA-seq and gene expression analysis were performed following themethods described previously in Sections 1.10 and 1.7.

Reagent: SYBR-Green (TOYOBO)

Primers for Q-PCR: Gapdh-F: CACTCTTCCACCTTCGATGC Gapdh-R:CTCTTGCTCAGTGTCCTTGC Igf2-F: CTAAGACTTGGATCCCAGAACC Igf2-R:GTTCTTCTCCTTGGGTTCTTTC Gtl2-F: TTGCACATTTCCTGTGGGAC Gtl2-R:AAGCACCATGAGCCACTAGG Dlk-F ACTTGCGTGGACCTGGAGAA Dlk-R:CTGTTGGTTGCGGCTACGAT H19-F: CATGTCTGGGCCTTTGAA H19-R: TTGGCTCCAGGATGATGT

Genome-wide methylation level analysis was performed following themethod as described in Section 1.11.

The results of Q-PCR showed that the expression of H19 and Gtl2 isdown-regulated while the expression of Igf2 and Dlk1 is up-regulated inDKO-AG-haESCs (Fig. S3D). The gene expression profiles of DKO-AG-haESCs,normal AG-haESCs and round spermatids were compared. Clustering databased on RNA-seq reveals that the expression profile of DKO-AG-haESCs ishighly similar to that of WT-AG-haESCs, but greatly different from thatof round spermatids (FIGS. 2F, S3E). Further analysis of the expressionprofiles of imprinted genes in DKO-AG-haESCs and WT-AG-haESCs revealedthat all imprinted genes in DKO-AG-haESCs and WT-AG-haESCs haveextremely similar expression levels (FIG. 2G). In order to furtherevaluate the epigenetics, reduced representation bisulfate sequencing(RRBS) was performed to detect the genome-wide methylation level. Asshown in FIGS. 2H, S3F, and S3G, knockouts of H19 and Gtl2 DMRs do notalter the methylation levels in the promoter regions of all the genesand imprinted genes detected. Taken together, the results show that H19and Gtl2 DMRs are two major impairments for AG-haESCs in obtainingcharacteristics resembling spherical spermatid.

Example 8

Production of Semi-Cloned Mice with DKO-AG-haESCs Carrying MultipleGenetic Modifications

A. Construction of DKO-AG-haESCs Carrying Multiple GeneticModifications:

The construction was carried out following the method as described inSection 1.4.

Initial cells: DKO-AG-haESCs. Knockouts of the TET family of genesemployed the DKO-AG-haESCs prepared in Example 4, and knockouts of thep53 family of genes employed the DKO-AG-haESCs prepared in Example 2.

Target mutations: mutations of Tet1, Tet2, Tet3, and p53 family

Construction procedure:

Construction procedure of Tet-TKO-DAH:

sgRNAs of Tet1, Tet2, and Tet3 were annealed respectively, and ligatedto a BbsI digested px330-mCherry plasmid respectively, and positiveplasmids in which sgRNAs were ligated were picked up by sequencing.

Tet1 sgRNA sequence: (SEQ ID NO: 5) GGCTGCTGTCAGGGAGCTCATet2 sgRNA sequence:  (SEQ ID NO: 6) GAAAGTGCCAACAGATATCCTet3 sgRNA sequence: (SEQ ID NO: 7) AAGGAGGGGAAGAGTTCTCG

The plasmids expressing the sgRNAs of Tet1, Tet2, and Tet3 wereco-transformed into the DKO-AG-haESC cell line. mCherry positive cellswere sorted, and plated in a Petri dish. After 5 days of growth, theclones were picked and passaged for amplification. The established cellline was identified for the Tet1, Tet2, and Tet3 mutations by sequencingthe PCR product.

Primers for sequencing: Tet1 check-F: GCCCCTGTTGTCTTATACGTTTet1 check-R: CATTCGCCTCAGGACCAC Tet2 check-F: CCGCCACAAGAAAATATGTCCTet2 check-R: AGCTAACTCTGGCAAACACC Tet3 check-F: CAGAGTGGCCTCAGTTTCCCTet3 check-R: ACAACTTTTACCCAGGAGTCACAC

Construction procedure of p53-TKO-DAH:

sgRNAs of p53, p63, and p73 were annealed respectively, and ligated to aBbsI digested px330-mCherry plasmid respectively, and positive plasmidsin which sgRNAs were ligated were picked up by sequencing.

p53 sgRNA sequence: (SEQ ID NO: 8) CACCTGGGCTTCCTGCAGTCp63 sgRNA sequence: (SEQ ID NO: 9) TGGGCCCGGGTAATCTGTGTp73 sgRNA sequence: (SEQ ID NO: 10) TGTCGATAGGAGTCAACCAA

The plasmids expressing the sgRNAs of p53, p63, and p73 wereco-transformed into the DKO-AG-haESCs cell line. mCherry positive cellswere sorted, and plated in a Petri dish. After 5 days of growth, theclones were picked and passaged for amplification. The established cellline was identified for the p53, p63, and p73 mutations by sequencingthe PCR product.

P53 check-F: CCCCTGTCATCTTTTGTCCCT P53 check-R: AAGAGAGTTCCACGTCCCCTGP63 check-F: CACACCAAATAATGCCAATT P63 check-R: CAGACTCTCTTACCGTCCAGP73 check-F: GACCCACTTCTAAACCTGCC P73 check-R: CCATACCTCCTGTGCTCCTG

B. Construction of Semi-Cloned Mice:

Following the method as described in Section 1.9, the cells constructedabove were used as a donor for ICAHCI to construct semi-cloned mice.

C. Results

In this example, Tet1, Tet2 and Tet3 were mutated in DKO-AG-haESCs byCRISPR-Cas9 method. The plasmids constructed to express Cas9 and 3sgRNAs of Tet1, 2, and 3 (FIG. 3A) (see Wang, H., Yang, H., Shivalila,C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., and Jaenisch, R. (2013).One-step generation of mice carrying mutations in multiple genes byCRISPR/Cas-mediated genome engineering. Cell 153, 910-918) weretransformed into DKO-AG-haESCs. 56 DKO-AG-haESC lines were finallyestablished. The sequencing of the PCR products of the Tet1, 2, and 3gene revealed that triple gene mutations occur in 18 cell lines(designated as Tet-TKO-DAH-1 through Tet-TKO-DAH-18) (FIGS. 3B-D andS4A). ICAHCI results of 4 of the cell lines indicated that SC mice withTet1, 2 and 3 mutations can be obtained with corresponding rate (FIGS.3E and S4B, Table 1 and Table S3). Corresponding SC mice can also beobtained with DKO-AG-haESCs with mutant p53 family of genes by ICAHCI(Figs. S4D, S4E, Table 1 and Table S3). These results show thatWT-AG-haESCs are failed to produce viable SC mice after in vitro geneticmanipulation, but DKO-AG-haESCs are still able to efficiently and stablyproduce semi-cloned mice after gene editing.

Example 9

Gene Editing of DKO-AG-haESCs and Production of Semi-Cloned MiceTherewith

A. Construction of Genetically Edited DKO-AG-haESC Cell Line

Initial cells: DKO-AG-haESCs (prepared in Example 4)

Construction procedure:

Construction procedure of Tet1&3-KI-DAH:

sgRNAs of Tet1, and Tet3 were annealed and ligated to a BbsI digestedpx330-mCherry plasmid respectively, and positive plasmids in whichsgRNAs were ligated were picked up by sequencing. For the preparation ofTet1-EGFP and Tet3-ECFP donors, pEGFP-N1 plasmid and pECFP-N1 plasmidwere respectively used as a template (primers: P2A-fluorescence F:GCCACGAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTGTGAGCAAGGGCG AGGAG

P2A-fluorescence R: CTTGTACAGCTCGTCCATG), a sequence encoding EGFP orECFP was amplified, and then correspondingly ligated to a multiclonalsite on a pMD19-T vector, to obtain a pMD19-T-EGFP/ECFP vector.Subsequently, the left and right homologous arms of the gene of interestwere respectively inserted into two sides of the EGFP or ECFP gene inthe pMD19-T-EGFP/ECFP vector.

Sequences of homologous arms:

TET1LA (left homologous arm): (SEQ ID NO: 11)

TET1 RA (right homologous arm): (SEQ ID NO: 12)

TET3 LA (left homologous arm): (SEQ ID NO: 13)

TET3 RA (right homologous arm): (SEQ ID NO: 14)

The 4 plasmids, comprising plasmids carrying Tet1 and Tet3 sgRNA, andTet1-EGFP and Tet3-ECFP donors were co-transfected into theDKO-AG-haESCs cell line. mCherry positive cells were sorted, and platedin a Petri dish. After 5 days of growth, the clones were picked andpassaged for amplification. The established cell line was identified forthe Tet1-EGFP and Tet3-ECFP knockins by PCR. The double knock-in cellswere designated as Tet1&3-KI-DAH.

Primers for identification by PCR: Tet1 LA-F: TTTGTGTCTATGAACTACCAGTGAGTet1 LA-F: CAGGCCCGGGGTTTTCTTC Tet1 RA-F: CAACGAGAAGCGCGATCACATet1 RA-F: TTTTGACTGATCCCAATTTGCCT Tet3 LA-F: TGTTCACTGGTGAAGGCCAGTet3 LA-F: GAACAGCTCCTCGCCCTTG Tet3 LA-F: TGAGCAAAGACCCCAACGAGTet3 RA-R: ATCGACAAACTTTGGGGCGA

Construction procedure of Tet-TKI-DAH:

sgRNA of Tet2 was annealed and ligated to a BbsI digested px330-mCherryplasmid, and a positive plasmid in which sgRNA was ligated was picked upby sequencing.

For the preparation of Tet2-mCherry donor, by using pmCherry-N1 as atemplate, and P2A-fluorescence F:GCCACGAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTGTGAGCAAGGGCG AGGAG andP2A-fluorescence R: CTTGTACAGCTCGTCCATG as primers, a sequence encodingmCherry was amplified and then ligated to a pMD19-T carrier.Subsequently, the left and right homologous arms of the gene of interestwere respectively inserted into two sides of the mCherry sequence in thepMD19-T-mCherry vector.

TET2 LA (left homologous arm): (SEQ ID NO: 15)

TET2 RA (right homologous arm): (SEQ ID NO: 16)

The 2 plasmids, comprising the plasmid carrying Tet2 sgRNA and theTet2-mCherry donor were co-transfected into the Tet1&3-KI-DAH cell line.mCherry positive cells were sorted, and plated in a Petri dish. After 5days of growth, the clones were picked and passaged for amplification.The established cell line was identified for the Tet2-mCherry knock-inby PCR. The positive cell clone was the Tet-TKI-DAH cell line.

Primers for identification: Tet2 LA-F: CACACCCTTCACCAACAGACG Tet2 LA-R:ATCTCGAACTCGTGGCCGTT Tet2 RA-F: AAGACCACCTACAAGGCCAAG Tet2 RA-R:GGTAGGCAAAGTGCTTTTCTAAGAC

B. Construction of Semi-Cloned Mice:

Following the method as described in Section 1.9, the cells constructedabove were used as a donor for ICAHCI to construct semi-cloned mice.

C. Results

In this example, DKO-AG-haESCs were obtained in which endogenous Tet1,Tet2 and Tet3 were knocked in different fluorescent reporter groups.First, DKO-AG-haESCs were transfected with a plasmid co-expressing Cas9and Tet1 and Tet3 sgRNAs (FIG. 3A) and a double stranded DNA donorvector having EGFP and ECFP reporter groups fused respectively to thelast terminator of Tet1 and Tet3 (FIGS. 3H and S5A). A total of 150DKO-AG-haESC cell lines were obtained, with 10 Tet1-EGFP knock-in and 7Tet3-ECFP knock-in cell lines (Figs. S5B and S5C). One of the cell linescarrying both Tet1-EGFP and 7 Tet3-ECFP knock-ins was designated as Tet1& 3-KI-DAH-1 (Figs. S5D and S5E). ICAHCI results showed thatDKO-AG-haESCs carrying Tet1-EGFP and Tet3-ECFP knock-ins have similarsemi-cloned mice production capability to WT DKO-AG-haESCs (Figs. S5Fand S5G, Table 1 and Table S3). Next, the plasmid expressing Cas9 andTet2 sgRNA and the double-stranded DNA donor vector having the mCherryreporter group fused to the last terminator of the Tet2 gene weretransfected into the Tet1 & 3-KI-DAH-1 cell line (FIGS. 3A and 3H). Outof the established 130 cell lines, 8 red-KFP knock-in haploid cell lineswere identified (designated as Tet-TKI-DAH-1 through Tet-TKI-DAH-8)(FIGS. 31 and S5H). Finally, the developmental potential of theTet-TKI-DAH cell line was verified by ICAHCI. Consistently, Tet-TKI-DAHwas able to efficiently produce viable semi-cloned mice by injectioninto oocytes (Figs. S5S, S5J, Table 1, and Table S3). Taken together,these results indicate that it is a possible route to produce SC micehaving corresponding genetic characteristics by ICAHCI after multi-genegenetic manipulation of DKO-AG-haESCs.

Example 10

Large-Scale Production of Heterozygous Mutant SC Mice with DKO-AG-haESCsCarrying sgRNA Library

A. Construction of DKO-AG-haESCs Carrying sgRNA Library

Initial cells: IG^(Δ) ^(DMR) -H19^(Δ) ^(DMR) -AGH-1

Following the method as described in Section 1.5, the virus was preparedand DKO-AG-haESCs were infected with a genome-wide lentiviral sgRNAlibrary, and then transfected with a pX330-mCherry plasmid expressingCas9, to finally construct DKO-AG-haESCs carrying the sgRNA library.

B. Construction of Semi-Cloned Mice:

Following the method as described in Section 1.9, semi-cloned mice wereconstructed using the previously constructed cells as a donor forICAHCI.

Detection of sgRNAs Carried in Semi-Cloned Mice:

The sgRNAs were detected by PCR amplification using specific primers,and then the PCR product was subjected to agar gel electrophoresis toobserve the presence of a strip.

Lenti-sg-F: GTTACTCGAGCCAAGGTCGG Lenti-sg-R: GACTCGGTGCCACTTTTTCA

Detection of Allelic Mutation:

According to the sgRNA sequencing, the corresponding gene inserted wasidentified, and then the upstream and downstream primers were designedby the conventional method in the vicinity of the target gene sgRNA,followed by PCR and sequencing.

C. Results

At present, the genome-wide sgRNA library has been successfullyestablished and used in the loss-of-function genetic screening in humanand mouse cells (Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-HerreraMdel, C., and Yusa, K. (2014). Genome-wide recessive genetic screeningin mammalian cells with a lentiviral CRISPR-guide RNA library. Naturebiotechnology 32, 267-273). This experiment demonstrates thatDKO-AG-haESCs are capable of carrying the sgRNA library and that a largenumber of mutant mouse models can be obtained simply by a one-stepmethod by ICAHCI (FIG. 4A) (this method is known as Lenti-sgRNA+pX330).In this test, a newly established and more definite mouse lentivirallibrary was used. This library had 87,897 sgRNAs designed for the genesof 19,150 encoded proteins in mice. 1.0×10⁷ haploid cell line IG^(Δ)^(DMR) -H19^(Δ) ^(DMR) -AGH-1 enriched by FACS were infected with thegenome-wide lentiviral sgRNA library. After 2 days, these infected cellswere treated with puromycin for 7 days, followed by transfection withthe pX330-mCherry plasmid expressing Cas9. Haploid cells expressingmCherry indicated the successful transfection and expression of Cas9,which were then used in ICAHCI experiments following FACS enrichment(FIG. 4B). To detect whether the haploid gene is induced to be mutatedby CRISPR-Cas9, 7 haploid cell clones were randomly selected. All of thedetected cell clones carried one sgRNA, which was also found to bemutated by DNA sequencing, indicating that gene mutations can besuccessfully induced in haploid cells by CRISPR-Cas9.

114 semi-cloned mice were obtained by three independent ICAHCIexperiments (FIG. 4E and Table 2), of which 82 carried one sgRNA (FIG.4F). Sequencing of the PCR products of sgRNA revealed that 43 SC miceharbored one allelic mutation of the gene of interest, 39 of whichcarried insertions or knockouts mutation (indel) that result inframeshift, leading to the loss of function of one allele (FIG. 4G).Interestingly, all the mutations were of the same genotype, indicatingthat gene mutations are realized in haploid cells after the transientexpression of Cas9. Although the remaining 39 mice carried one sgRNA, noDNA cleavage was shown at the site needed to be mutated. This rate wasconsistent with that previously observed in human cells (Zhou, Y, Zhu,S., Cai, C., Yuan, P., Li, C., Huang, Y., and Wei, W. (2014).High-throughput screening of a CRISPR/Cas9 library for functionalgenomics in human cells. Nature 509, 487-491) or mouse embryos (Wu, Y.,Liang, D., Wang, Y., Bai, M., Tang, W., Bao, S., Yan, Z., Li, D., andLi, J. (2013). Correction of a genetic disease in mouse via use ofCRISPR-Cas9. Cell Stem Cell 13, 659-662). This data demonstrates thatthe introduction of genetic mutations into SC mice can be achieved byICAHCI of DKO-AG-haESCs harboring the sgRNA library that are transientlytransfected to express Cas9, to finally obtain a large number ofheterozygous mutant mice in one step.

Example 11

Production of Mice Harboring sgRNA-Mediated Biallelic Mutations withDKO-AG-haESCs in One Step

A. Construction of sgRNA-Bearing Haploid Cells

Initial cells: IG^(Δ) ^(DMR) -H19^(Δ) ^(DMR) -AGH-1

DKO-AG-haESCs were infected with the lentiviral CRISPR-sgRNA libraryfollowing the method as described in Section 1.5, to obtain a cell linecarrying the lentiviral sgRNA library.

B. Construction of Semi-Cloned Mice:

The nuclei of the haploid cells carrying sgRNA were injected into matureoocytes by ICAHCI, followed by intracytoplasmic injection of Cas9 mRNAinto the reconstructed oocyte (where this protocol was known asLenti-sgRNA+Cas9 injection). Semi-cloned mice were then constructed byembryo transfer.

sgRNA detection in semi-cloned mice: The detection was carried outfollowing the method as described in Example 9.

Detection of allelic mutation: The detection was carried out followingthe method as described in Example 9. In addition, the PCR product ofthe target gene was ligated into the pMD19-T vector, and then the cloneswere picked up and sequenced.

C. Results

In this experiment, haploid cells carrying sgRNA were injected intomature oocytes by ICAHCI and then Cas9 was injected into thereconstructed oocytes (where this protocol was known as Lenti-sgRNA+Cas9injection). A total of 45 SC mice carrying one sgRNA were born, of which22 had genetic mutations (Table 2). Sequencing of the PCR product of thegene of interest revealed that 10 mice had only a monoallelicmodification, and other 12 mice were biallelic mutant (which is 23.5% ofall born SC mice). Sequencing by TA cloning of the 7 biallelic mutant SCmice revealed that approximately 63% of the clones carried the insertionor deletion mutations (Table S4).

Example 12

Production of Mice Harboring sgRNA-Mediated Biallelic Mutations withDKO-AG-haESCs in One Step

A. The sgRNA-bearing haploid cells were constructed following the methodas described in Example 10A and the pX330-mCherry plasmid was thentransiently transfected into the sgRNA-bearing haploid cells.

B. Construction of Semi-Cloned Mice:

The nuclei of the haploid cells obtained in A were injected into oocytesby ICAHCI, followed by intracytoplasmic injection of Cas9 mRNA.Semi-cloned mice were then constructed by embryo transfer.

sgRNA detection in semi-cloned mice: The detection was carried outfollowing the method as described in Example 9.

Detection of allelic mutation: The detection was carried out followingthe method as described in Example 11.

C. Results

In this experiment, the pX330-mCheny plasmid was transiently transfectedinto the sgRNA-bearing haploid cells, and then the cells were injectedinto oocytes, followed by injection of Cas9 mRNA (Fig. S6A and S6B)(where this protocol was known as Lenti-sgRNA+pX330+Cas9 injection). Atotal of 27 SC mice carrying one sgRNA were obtained. 22 mice carriedgenetic mutations (Figs. S6C and S6D and Table 2), in which 13 of themutant mice carried biallelic mutations (Fig. S6E) (which was 41.9% ofall the SC mice) and the other 9 mice were monoallelic mutant. TAcloning and sequencing results of 5 biallelic mutant mice revealed thatabout 79% of the clones have insertion or deletion mutations (Figs. S6Fand S6G and Table S4)

Example 13

Production of Mice Harboring sgRNA-Mediated Biallelic Mutations withDKO-AG-haESCs in One Step

A. Construction of DKO-AG-haESCs with Constant Expression of Cas9 andsgRNA Library

Initial cells: IG^(Δ) ^(DMR) -H19^(Δ) ^(DMR) -AGH-1

Specific construction of cell lines by Lenti-Cas9+lenti-sgRNA method:The construction was carried out following the method “co-infection withlentiviral Cas9+lentiviral sgRNA library” as described in Section 1.5.

The cell line integrated with lentiviral Cas9 and lentiviral sgRNAlibrary was further sorted by FACS to enrich the haploid cells for usein subsequent ICAHCI operation.

B. Construction of Semi-Cloned Mice:

Following the method as described in Section 1.9, semi-cloned mice wereconstructed by ICAHCI using DKO-AG-haESCs with constant expression ofCas9 and sgRNA library as a donor.

Detection of allelic mutation: The detection was carried out followingthe method as described in Example 11.

Exemplary primers for identifying the mutations in mice:

Polm check-F: TCCGATGGGAAGCCAAAAGC Polm check-R: CGTACCGCAACCGCGAAGTAScube1 check-F: CCATAATAATCCACTTCCAT Scube1 check-R:CCAACCCCTGTCCACTACCT

C. Results

In this experiment, DKO-AG-haESCs with constant expression of Cas9 andsgRNA library were obtained by two rounds of screening with drugs. SCmice were then generated by ICAHCI (FIG. 5A-D) (where this protocol wasknown as Lenti-Cas9+lenti-sgRNA). 272 (18.7%) of the 1,453 embryosreconstructed by ICAHCI were developed fully (FIG. 5E) and the birthrate was basically similar to that obtained with WT DKO-AG-haESCs orDKO-AG-haESCs carrying different gene modifications (Table 2),indicating that after multiple genetic manipulations, the ability ofDKO-AG-haESCs to produce SC mice is not affected. A total of 224 SC micecarried one sgRNA and were used for subsequent genotyping (FIG. 5F andTable 2). The results showed that 143 SC mice are genetically mutated,of which 83 are biallelic mutant mice (FIG. 5G). 60 monoallelic mutantSC mice were obtained. Although Cas9 in these mice was supposed to beconstantly expressed, silencing of the viral vector led totranscriptional silencing of Cas9 at different development stages of theSC mice. TA cloning and sequencing of 26 biallelic mutant SC mice showedthat about 66.3% of the clones have insertion or deletion mutations(FIGS. 5H and I). In order to further determine the mutation efficiencythroughout the body of the SC mice, the genetic mutations in differentorgans (comprising the brain, heart, kidney, liver and lung) of 4 SCmice were analyzed and all of these organs were found to have biallelicmutations. Finally, TA cloning and sequencing of different organs in oneSC mouse carrying Scube1 mutation was performed. The results showed thatabout 80% of the clones have insertion or deletion mutations (FIG. 5J).Interestingly, this SC mouse died within an hour after birth, consistentwith previous reports that the Scube1 mutant mouse died shortly afterbirth (Tu, C. F., Yan, Y. T., Wu, S. Y., Djoko, B., Tsai, M. T., Cheng,C. J., and Yang, R. B. (2008). Domain and functional analysis of a novelplatelet-endothelial cell surface protein, SCUBE1. The Journal ofbiological chemistry 283, 12478-12488). In conclusion, the aboveexperimental results provide sufficient evidence for the possibility ofobtaining a large number of mutant mice with DKO-AG-haESCs carrying thesgRNA library, thereby achieving the large-scale gene knockout-basedscreening at the mouse level, which greatly simplifies theDKO-AG-haESC-mediated gene knockout-based screening.

TABLE 1 Summary of in-vivo development of ICAHCI embryos Number ofgrowth-arrested Number of Number mice (% of normal mice Cell of numberof (% of number Type of donor Haploid ES cell passage embryos embryos ofembryos cells line number transferred transferred) transferred) SingleDMR KO H19^(Δ) ^(DMR) -AGH p8-pl7 1443 39 (2.7)  86 (5.9)^(c) AG-haESCsCells IG^(Δ) ^(DMR) -AGU p8-p23  499 12 (2.4)   4 (0.8)^(d) CellsDKO-AG- H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) - pl9-p33  939  4 (0.4) 210 (22.4)haESCs AGH Cells IG^(Δ) ^(DMR) -H19^(Δ) ^(DMR) - p24-p28  544  5 (0.9)105 (19.3) AGH Cells H19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) - p26-p37  510  1(0.2)  87 (17.1) AGH-OG3 Cells Subtotal pl9-p37 1993 10 (0.5) 402(20.2)^(e) DKO-AG-ha Tet-TKO-DAH p35-p37  407  4 (1)  59 (14.5) ESCscarrying Cells Tel 1, 2 and 3 triple mutations DKO-AG-ha p53-TKO-DAHp41-p46  660  2 (0.3) 111 (16.7) ESCs carrying Cells p53, 63 and 73triple mutations DKO-AG-ha Tet1&3-KI- p40-p47  138  1 (0.7)  21 (15.2)ESCs carrying DAH-1 Tet1 and 3 knock-ins DKO-AG-ha Tet-TKI-DAH-1 p47-p51 874  6 (0.7) 151 (17.3) ESCs carrying Tet1, 2 and 3 knock-ins WTAG-haESCs AGH Cells^(a) p9-p20  294  3 (1.0)   2 (0.7) AGH-OG-3 pl2-p26 379  6 (1.6)   7 (1.8) Cells^(b) Round spermatid  125 0  28 (22.4)^(a)AG-haESCs: prepared according to the present invention. See Method1.3. ^(b)AG-haESCs: derived from the prior art, supplied by the authorof (Yang, H., Shi, L., Wang, B. A., Liang, D., Zhong, C., Liu, W., Nie,Y., Liu, J., Zhao, J., Gao, X., et al. (2012). Generation of geneticallymodified mice by oocyte injection of AG-haESCss. Cell 149, 605-617).^(c)For H19^(Δ) ^(DMR) -AGR Cells, p < 0.05, compared with WT AG-haESCs^(d)For IG^(Δ) ^(DMR) -AGU Cells, p > 0.05, compared with WT AG-haESCs^(e)For DKO-AG-haESCs, p < 0.001, compared with WT AG-haESCs

TABLE SI Summary of in-vivo development of ICAHCI embryos constructedwith different AG-haESCs Number of growth-arrested Number of Number mice(% of normal mice Cell of number of (% of number Type of donor HaploidES cell passage embryos embryos of embryos cells line number transferredtransferred) transferred) H19-DMR H19^(Δ) ^(DMR) -AGH-1 p8 375 10 (2.7)10 (2.7) KO p9 180  9 (5.0) 13 (7.2) AG-haESCs H19^(Δ) ^(DMR) - AGH-2 p8210  5 (2.4)  6 (2.9) p9 116  2 (1.7)  4 (3.4) p11 123  3 (2.4)  3 (2.4)H19^(Δ) ^(DMR) -AGH-3 p8 271  1 (0.4) 29 (10.7) p17 168  9 (5.4) 21(12.5) IG-DMR KO IG^(Δ) ^(DMR) -AGH-1 p14  84  0  0 AG-haESCs p15 100  4(4)  0 p17  54  4 (7.4)  1 (1.9) p23  81  1 (1.2)  0 IG^(Δ) ^(DMR)-AGH-2 p8 180  3 (1.7)  3 (1.7) H19^(Δ) ^(DMR) - H19^(Δ) ^(DMR) -IG^(Δ)^(DMR) - pl9 175  1 (0.6) 44 (25.1) IG^(Δ) ^(DMR) -AGH AGH-1   CellsH19^(Δ) ^(DMR) -IG^(Δ) ^(DMR) - pl9 245  2 (0.8) 56 (22.9) AGH-2 H19^(Δ)^(DMR) -IG^(Δ) ^(DMR) - p29 204  0 46 (22.5) AGH-3 H19^(Δ) ^(DMR)-IG^(Δ) ^(DMR) - p29 165  1 (0.6) 32 (19.4) AGH-4 p33 150  0 32 (21.3)IG^(Δ) ^(DMR) -H19^(Δ) ^(DMR) - IG^(Δ) ^(DMR) -H19^(Δ) ^(DMR) - p24 120 1 (0.8) 26 (21.7) AGH Cells AGH-1 IG^(Δ) ^(DMR) -H19^(Δ) ^(DMR) - p24180  2 (1.1) 47 (26.1) AGH-2 p28 244  2 (1.3) 32 (22.2) IG^(Δ) ^(DMR)-H19^(Δ) ^(DMR) - IG^(Δ) ^(DMR) -H19^(Δ) ^(DMR) - p26 118  0 17 (14.4)AGH-OG3 AGH-OG3-1 Cells IG^(Δ) ^(DMR) -H19^(Δ) ^(DMR) - p30 192  1 (0.5)36 (18.8) AGH-OG3-2 p37 200  0 34 (17) WT AGH-OG-3^(a) p12  82  0  1(1.2) AG-haESCs p20  93  4 (4.3)  0 p24 140  2 (1.4)  5 (3.6) p26  64  0 1 (0.9) AGH-2^(b) p19 114  0  1 (0.9) p20  62  3 (4.8)  0 AGH-3^(c) p9118  0  1 (1.2) ^(a)AG-haESCs: made according to the prior art.^(b)AG-haESCs: derived from the prior art (Yang, H., Shi, L., Wang, B.A., Liang, D., Zhong, C., Liu, W., Nie, Y., Liu, J., Zhao, J., Gao, X.,et al. (2012). Generation of genetically modified mice by oocyteinjection of androgenetic haploid embryonic stem cells. Cell 149,605-617)

TABLE S3 Summary of in-vivo development of ICAHCI embryos constructedwith genetically modified DKO-AG-haESCs Number of growth-arrested Numberof Number mice (% of normal mice Cell of number (% of number Type ofdonor Haploid ES passage embryos of embryos of embryos cells cell linenumber transferred transferred) transferred) DKO-AG-ha ESCsTet-TKO-DAH-1 p37 131 2 (1.5) 17 (13) Carrying Tet1, 2 Tet-TKO-DAH-2 p37 54 0  9 (16.7) and 3 Mutations Tet-TKO-DAH-3 p36 174 1 (0.6) 27 (15.5)Tet-TKO-DAH-4 p35  48 1 (2.1)  6 (12.5) DKO-AG-ha p53-TKO-DAH-l p42 1821 (0.5) 34 (18.7) ESCs carrying p53, p44 186 0 32 (17.2) p63 and p73p53-TKO-DAH-2 p41 154 0 23 (14.9) mutations p42  48 1 (2.1)  8 (16.7)p53-TKO-DAH-3 p46  90 0 14 (15.6) DKO-AG-haESCs Tet1&3-KI- p40  58 1(1.7) 10 (17.2) carrying Tet1 and 3 DAH-1 p47  80 0 11 (13.8) knock-insDKO-AG-ha ESCs Tet-TKI-D p47  48 1 (2.1)  8 (16.7) carrying Tet1, 2 AH-1p49 174 0 32 (19) and 3 knock-ins Tet-TKI-D p47  96 3 (3) 10 (10.4) AH-2p50 136 1 (0.7) 22 (16.2) Tet-TKI-D AH-3 p49  48 0  8 (16.7) Tet-TKI-DAH-4 p50 102 1 (1.0) 14 (13.7) Tet-TKI-D AH-5 p51 174 0 39 (22.4)Tet-TKI-D AH-6 p51  96 0 18 (18.8)

TABLE 2 Summary of in-vivo development of ICAHCI embryos constructedwith DKO-AG-haESCs carrying sgRNA library Number of SC Number NumberNumber Number of Number of mice of SC of SC of SC SC mice SC mice Numberof (% of number mice mice with mice with with with embryos of embryoswithout sgRNA one biallelic monoallelic Strategy transferredtransferred) sgRNA (n > 2) sgRNA mutation mutation Lenti- 580 114 (19.7)11 21 82 0 43 sgRNA+ pX330 Lenti- 306  51 (16.7) 0 4 47 12 10 sgRNA+Cas9 injection Lenti-sgR 238  31 (13.5) 3 1 27 13 9 NA+pX330+ Cas9injection Lenti-Cas 1453 272 (18.7) 22 13 237 83 60 9+lenti- sgRNA

The above examples are provided for the purpose of illustrating theembodiments disclosed in the present invention and are not to beconstrued as limiting the present invention. In addition, variousmodifications and variations of the methods and compositions madewithout departing from the scope and spirit of the present invention areapparent to those skilled in the art. While the present invention hasbeen specifically described in connection with various specificpreferred embodiments thereof, it is to be understood that the presentinvention is not limited to these specific embodiments. In fact, variousapparent modifications made to the present invention as described aboveare embraced in the scope of the present.

Sequence listing <110>Shanghai Institutes for Biological Sciences Chinese Academy of Sciences<120>Androgenetic haploid embryonic stem cell (AG-haESC), and preparationmethod and use thereof <130> 140709 <160> 16 <170> PatentIn version 3.3<210> 1 <211> 20 <212> DNA <213> Artificial <220> <223>IG-DMR-sgRNA1 sequence <400> 1 cgtacagagc tccatggcac 20 <210> 2 <211> 20<212> DNA <213> Artificial <220> <223> IG-DMR-sgRNA2 sequence <400> 2ctgcttagag gtactacgct 20 <210> 3 <211> 20 <212> DNA <213> Artificial<220> <223> H19-3.8K sgRNA-1 sequence <400> 3 catgaactca gaagagactg 20<210> 4 <211> 20 <212> DNA <213> Artificial <220> <223>H19-3.8K sgRNA-2 sequence <400> 4 aggtgagaac cactgctgag 20 <210> 5 <211>20 <212> DNA <213> Artificial <220> <223> Tet1 sgRNA sequence <400> 5ggctgctgtc agggagctca 20 <210> 6 <211> 20 <212> DNA <213> Artificial<220> <223> Tet2 sgRNA sequence <400> 6 gaaagtgcca acagatatcc 20 <210> 7<211> 20 <212> DNA <213> Artificial <220> <223> Tet3 sgRNA sequence<400> 7 aaggagggga agagttctcg 20 <210> 8 <211> 20 <212> DNA <213>Artificial <220> <223> p63 sgRNA sequence <400> 8cacctgggct tcctgcagtc 20 <210> 9 <211> 20 <212> DNA <213> Artificial<220> <223> p63 sgRNA sequence <400> 9 tgggcccggg taatctgtgt 20 <210> 10<211> 20 <212> DNA <213> Artificial <220> <223> p73 sgRNA sequence <400>10 tgtcgatagg agtcaaccaa 20 <210> 11 <211> 895 <212> DNA <213>Artificial <220> <223> Sequence of TET1 LA left homologous arm <400> 11aagcttaagg ttcacacaac actaagagct tttcatcagc ctcatctact tctcacctag  60tgaaagacga atctacagac ttctgtcccc tgcaggcttc ctccgcagaa acatctacct 120gtacgtacag taaaacagcc tcaggtgggt ttgcagaaac aagtagtatt ctccactgca 180caatgccttc tggagcacac agtggtgcta atgcagctgc tggggaatgt actggaacgg 240tgcagcctgc cgaggtggct gctcatcctc accagtctct tcccacagcc gattctcccg 300ttcatgctga gcctctcact agtccatctg agcagctaac ttctaaccag tcaaaccagc 360agctccctct cctcagcaat tctcagaaac tggcttcctg tcaggtggaa gatgagcggc 420accctgaagc ggatgagcct cagcaccccg aggacgataa cttgcctcaa cttgatgaat 480tctggtcaga cagtgaggag atctacgccg atccttcctt tggtggcgtg gcgatagcac 540ccattcacgg ctcggtgctc attgagtgcg ctcggaagga gettcatgct accacctctt 600tgcgctcccc caaacgaggg gtcccttttc gtgtgtccct tgtattctac cagcacaaaa 660gcctaaacaa gcctaatcat ggttttgata tcaacaaaat taagtgtaaa tgcaaaaaag 720taacgaaaaa aaagcccgca gaccgggagt gtcctgatgt atcccccgaa gccaatttat 780cacaccaaat tccttctcga gttgcatcaa ccttaacccg agacaatgtt gttaccgtgt 840ccccatactc tctcactcat gttgcgggac cctacaatcg ttgggtcgcg tcgac 895 <210>12 <211> 803 <212> DNA <213> Artificial <220> <223>Sequence of TET1 RA right homologous arm <400> 12taaaggcttc tctcatgtaa tgcctttgct aatgtggtgt agtgggtatt tttgtttgtt  60tgtttgtttt cttttgtttt tttgtttttt ccggtgctgt taaaaagaaa gtcattctgt 120tgtttactgt agctttgttt cgcccatttc aactccgacg taaatattaa aaaaaaaaaa 180agggtgaata cttaactgtg attacatttt gagaattggt agaaggtgaa cattttcagc 240aaaaataaac tttttatagt tttaaatact taaaggaaca tcttggttag gtgttggcca 300tgctagaacc atagagtctg gtgctttccc ccgggtttgt ttactattca gagggtttat 360aacaggttcc tgcaataaga agtaaagacc aagatgtagt gttaactcta cacagttcct 420ggtgctttaa ccacatcaac acacggagtg atgagctgag tgattgtttt ctggtgccat 480tgctcaagcc tcttccaatc attgccatcg tgtctgcaca tttctttgaa gtaaaccaat 540gaaatgcttt ttctcttaaa acatttctcc tatataaagt agttctctat tctcatgatg 600gttggaagct gttcgctaac tataaatgta tatattttaa aaagcacttt ttacttttaa 660gagtaacttg aaatagtata gtagcagaat cctattgtct attatgtgtg catatttgaa 720taccagagaa gtcatttgtt cttgctctgt agagtcccat cccgttaacc tcagcctgta 780ctcaaataac acacggcttc tgt 803 <210> 13 <211> 809 <212> DNA <213>Artificial <220> <223> Sequence of TET3 LA left homologous arm <400> 13ctggtcccag cctaactgag aagtcatggg ggatgggaac cggggatttc aacctcgccc  60tgaaaggtgg acctgggttc caagacaagt tgtggaatcc tgtgaaggtg gaggagggca 120ggattcccat accgggggcc aactcgctag acaaagcctg gcaagccttt ggcatgccct 180tgagctccaa cgagaagcta tttggggccc tgaagtcaga ggagaaactg tgggatccct 240tcagcctgga ggaggggaca gctgaggagc cccccagcaa gggggtggtg aaggaagaga 300agagtggacc cacagtgaaa gaggacgagg aggaactgtg gtcggacagt gaacacaact 360tcctggatga gaacataggc ggggtggccg tggccctcgc ccattgctcc atcctcatcg 420agtgtgcccg gcgagagctg catgccacca ctccactcaa aaaacccaac cgctgccacc 480ccacccgcat ctcgctggtc ttctaccaac acaagaacct caaccagtcc aaccacgggc 540tggcgctctg ggaggccaag atgaagcagc tggcggaatg ggcgcggcag cggcaagagg 600aggccgcacg cctgggcctg ggccagcagg aggccaagct ctacgggaag aagcgaaaat 660gggggggtgc tatggtggct gagccccagc aeaaagaaaa gaagggggct atccctaccc 720ggcaggcgct ggccatgccc acagacttcg cggtcaccgt gtcctcttac gcctacacaa 780aggtcactgg cccctacagc cgctggatc 809 <210> 14 <211> 837 <212> DNA <213>Artificial <220> <223> Sequence of TET3 RA right homologous arm <400> 14taggtgccag gagccagcgt acctcagcgt cgggcccggc ccgagctgct ctgtggtgct  60tttgccctca tgcccggggg cgggttgggg gtgcagaagt ctctctatat ctacatatat 120ggatatgctt atcatatata tgtatttatg gtccaaacct cagaactgac ccgccctccc 180tcctcttctt ccccagcact ttgaagaaac tacggctgtc gggtgatttt tttttttttt 240tgatcttaat atttatatct ccacgtttta tttttcccct tgttttgagg gggcttttat 300tttccttttg tttttaaaac tttatccttg tatatcacaa taatggaaag aaagtttata 360gtgtcctttc acaaaggagc gtagttttaa aattccattt aaaatatgta tttattgggt 420ttttttaaag caacaatagt aatgggttac aggtgggcag gaaaggccgg cagtgcttcc 480tgcctggcaa gcccagcctc ctgggggctg caggctctct cagccatctg cctcacaaac 540caaggttagc atgtagccct ggtttatccc tccttcccac aggctgggga gccttttggg 600accaccccgt tctcctctgt ggaatggggg aagctgcaga catctggggg cctggcctgg 660aaatgctggt gttagatatc cccagaaacc aggttggaag tagacagctt caagcttgct 720agtctccaca ctgaatcctc tctgtccgtt atttaccaag tcatatgatg tcctggttcg 780ctaggcagca cctcaagctg gagcaggagt cgagaggctc cgaggagctc ttctcgt 837 <210>15 <211> 824 <212> DNA <213> Artificial <220> <223>Sequence of TET2 LA left homologous arm <400> 15caaatgtaca ccacctagca acgttttctc cttaccccac coccaagatg gatagtcatt  60tcatgggagc tgcctccaga tcaccataca gccacccaca cactgactac aaaaccagtg 120agcatcatct accctctcac acgatctaca gctacacggc agcagcttcg gggagcagtt 180ccagccacgc cttccacaac aaggagaatg acaacatagc caatgggctc tcaagagtgc 240ttccagggtt taatcatgat agaactgctt ctgcccaaga actattatac agtctgactg 300gcagcagtca ggagaagcag cctgaggtgt caggccagga tgcagctgct gtgcaggaaa 360ttgagtattg gtcagatagt gagcacaact ttcaggatcc ttgcattgga ggggtggcta 420tagctccaac tcatgggtca attcttattg agtgtgcaaa gtgtgaggtt catgccacaa 480ccaaagtaaa cgatcccgac cggaatcacc ccaccaggat ctcacttgta ctgtataggc 540ataagaattt gtttctacca aaacattgtt tggctctctg ggaagccaaa atggctgaaa 600aggcccggaa agaggaagag tgcggaaaga atggatcaga ccacgtgtct cagaaaaatc 660atggcaaaca ggaaaagcgt gagcccacag ggccacagga acccagttac ctgcgtttca 720tccagtctct tgctgagaac acagggtctg tgactacgga ttctaccgtg actacatcac 780catatgcttt cactcaggtc acagggcctt acaacacatt tgta 824 <210> 16 <211> 792<212> DNA <213> Artificial <220> <223>Sequence of TET2 RA right homologous arm <400> 16tgacgctggc cattaggcca gaccaccaag gacgacctgt gagcagtatg tctttcatgg  60catgggccgt agggacaggt cacagcatct gtgacaaatg cagtgtgtgt ttgtgtgtat 120gtttattggg ggggggctgt cagctcacca gcaaaatagt ttattttatc attatattta 180atctctcccg tggtccatgg tggcattcag gaagagcatc ctatgcaagg gcacagtggg 240aaggaagcgc tggacatttg tcttgaaaac cactggttct cttattggct gaggtcatgc 300gtgtgccatg cccctcagca ctctacacgt aactgcttct agtattcagc gtgtgtaacc 360gtgggacaca gcgctgtagt agagcagttg caggatcatc tggtgctgac gtatgatgta 420ctgaagaaat actggaacta agacttttta acatgcaggt tttttactgt aatcttaata 480acttatttat caaagtagct acagaaagct taagtgaata atggcaaaac actgaatctg 540tttgggtgtt aacattaaat ggtgctacaa atggtgtttt taatagctga aaaatcaatg 600ccttctatca tctagccagt gtggtcgagg gccctggagg cactggggta cctctgattt 660tacatttcta tcttaattat tcagcttagt ttttaaaatg tggacatttc aaaggcctct 720ggattgtagt tatccaccga tgtccttgta ggactataat gtatagatat gcacacttac 780acatgtgtac tg 792

1. An androgenetic haploid embryonic stem cell (AG-haESC), in which H19DMR and IG-DMR are knocked out.
 2. The AG-haESC according to claim 1,wherein the AG-haESC is derived from a mammal, preferably from a rodent,more preferably from a murine, and most preferably from a mouse.
 3. Amethod for producing an AG-haESC, comprising: knocking out H19 DMR andIG-DMR from an AG-haESC, to obtain the AG-haESC.
 4. The method forproducing an AG-haESC according to claim 3, wherein the H19 DMR andIG-DMR are knocked out by CRISPR/Cas9-mediated genetic manipulation. 5.The method for producing an AG-haESC according to claim 3, furthercomprising: modifying one or more target genes of interest in theAG-haESC.
 6. (canceled)
 7. A method for constructing a geneticallymodified semi-cloned animal, comprising: combining an AG-haESC in whichH19 DMR and IG-DMR are both knocked out with an oocyte to obtain asemi-cloned embryo, and incubating the semi-cloned embryo to obtain asemi-clone animal.
 8. The method for constructing a genetically modifiedsemi-cloned animal according to claim 7, wherein the semi-cloned embryois obtained by ICAHCI with the AG-haESC in which H19 DMR and IG-DMR areboth knocked out as a donor for ICAHCI.
 9. The method for constructing agenetically modified semi-cloned animal according to claim 7, whereinone or more target genes of interest in the AG-haESC in which H19 DMRand IG-DMR are both knocked out are modified.
 10. The method forconstructing a genetically modified semi-cloned animal according toclaim 7, wherein the semi-cloned animal is a non-human mammal,preferably a rodent, more preferably a murine, and most preferably amouse.
 11. A genetically modified animal, which is a semi-cloned animalconstructed according to the method as set forth in claim 7, or asexually reproduced offspring of a semi-cloned animal constructedaccording to the method as set forth in claim
 7. 12. A method forconstructing a genetically modified semi-cloned animal library, which isone of Method 1, comprising the steps of: 1) infecting the AG-haESCsaccording to claim 1 with virus particles prepared with the lentiviralsgRNA library plasmids, to obtain an AG-haESC library carrying the sgRNAlibrary; 2) obtaining a semi-cloned embryo library by ICAHCI using avector expressing Cas9 and/or Cas9 mRNA, with AG-haESCs in the AG-haESClibrary carrying the sgRNA library as a donor for ICAHCI; and 3)incubating the embryos in the semi-cloned embryo library to obtain asemi-cloned animal library; and Method 2, comprising the steps of: 1)infecting the AG-haESCs according to claim 1 with lentiviral particlesexpressing Cas9 and lentiviral particles prepared with the lentiviralsgRNA library, to obtain an AG-haESC library with constant expression ofthe sgRNA library and Cas9; 2) obtaining a semi-cloned embryo library byICAHCI with AG-haESCs in the AG-haESC library with constant expressionof the sgRNA library and Cas9 as a donor for ICAHCI; and 3) incubatingthe embryos in the semi-cloned embryo library to obtain a semi-clonedanimal library.
 13. The method for constructing a genetically modifiedsemi-cloned animal library according to claim 12, wherein Step 2) inMethod 1 is selected from any one of: Method A, comprising: furthertransfecting the AG-haESC library carrying the sgRNA library with aplasmid expressing Cas9, and obtaining the semi-cloned embryos by ICAHCIusing the resultant AG-haESCs as a donor for ICAHCI; Method B,comprising: injecting AG-haESCs in the AG-haESC library carrying thesgRNA library, as a donor for ICAHCI, into mature oocytes by ICAHCI, andthen injecting Cas9 mRNA into the reconstructed oocytes, to obtain thesemi-cloned embryos; and Method C, comprising: further transfecting theAG-haESC library carrying the sgRNA library with a plasmid expressingCas9, injecting the resultant AG-haESCs, as a donor for ICAHCI, intomature oocytes by ICAHCI, and then injecting Cas9 mRNA into thereconstructed oocytes, to obtain the semi-cloned embryos.
 14. The methodfor constructing a genetically modified semi-cloned animal libraryaccording to claim 13, wherein the plasmid expressing Cas9 ispX330-mCherry plasmid.
 15. The method for constructing a geneticallymodified semi-cloned animal library according to claim 12, wherein theanimals in the semi-cloned animal library are heterozygous and/orbiallelic mutant animals.
 16. The method for constructing a geneticallymodified semi-cloned animal library according to claim 12, wherein thesemi-cloned animal library is a non-human mammalian library, preferablya rodent library, more preferably a murine library, and most preferablya mouse library.
 17. A genetically modified semi-cloned animal library,constructed according to the method for constructing a geneticallymodified semi-cloned animal library as set forth in claim 12.